By Suanne J. Klahorst
Enzymes protein molecules with catalytic functions are among the most unique and natural processing aids used in the food and beverage industry. In fact, the manufacture of many of todays processed fruits and vegetables, wines, and juices would not be possible without the use of enzymes. Not only do they function as catalysts for many important reactions; they meet the natural criteria by virtue of being made by living organisms.
Even with the understanding we have of genomics and how proteins are coded for and expressed in cells, we still have to rely on the cellular systems of living organisms to build a host of complex enzyme molecules cheaply and efficiently. All organisms digest, metabolize and grow through the reactions of a nearly incomprehensible number of specific enzymes. Most raw foods contain native, or endogenous, enzymes that are inactivated when heated or during processing. Strong acids, such as stomach acid, can also inactivate enzymes in raw foods, though some enzymes may survive the digestive tract.
Most commercial enzyme preparations come from bacterial
or fungal fermentation although some are extracted from plant
or animal parts. A common plant-derived enzyme is papain, a protein-degrading
enzyme from papaya that was once a common ingredient in meat tenderizers,
and is still used in brewing and other protein-modification applications.
Chymosin, a widely used animal enzyme, is a specific type of rennet
(sometimes called rennin) that is a very selective clotting protease
extracted from calf digestive organs. Chymosin is marketed for production
of cheese curd, and several types of traditional cheese cannot be duplicated
Enzymes have a positive reputation associated with health.
Active enzymes are often taken as dietary supplements to aid digestion.
An example of a supplement used in tablet form is lactase, which aids
lactose digestion and is useful to lactose-intolerant individuals.
Enzymes are specifically designed for their target molecules
their substrates by virtue of their shape, size, and the
chemical charges that precisely fit and bond to the substrate molecule.
The section of the enzyme that binds the substrate is called the active
site. An advantage of using enzymes is that they are specific and do
not interact with other components in the food or beverage. Another
advantage is that by catalyzing the same reactions repeatedly as long
as the substrate is available (referred to as turnover), they can be
used at very low concentration. Eventually, as the end products of the
enzyme reaction increase in concentration, the reaction is temporarily
inhibited by feedback inhibition. For example, if an enzyme catalyzes
the breakdown of polymer into glucose, high levels of glucose will likely
begin to inhibit the enzyme activity.
Most enzymes added in fruit processing cleave bonds the way that ripening enzymes do; however, in the case of processing, the ripening occurs at a faster rate and in a tank. With a few exceptions, commercial enzymes for fruit and vegetable processing are of the hydrolytic variety, reducing the molecular size of plant and animal constituents. Fruit-processing enzymes are often produced by fungal organisms and are not as heat-stable as the bacterial amylases used in high fructose corn syrup production, which can withstand temperatures of 100º C. Pasteurization temperatures or other heat treatments during packaging denature commercial fruit- and vegetable-processing enzymes. Denatured fruit-processing enzymes only contribute a miniscule amount to the total protein content of the food; hence they fall more under the category of processing aids than of food additives.
The enzyme for all seasons
Fruit and wine processors have relied mainly on enzymes produced by the fungal organism Aspergillus niger and its subspecies Aspergillus aculeatus. The most indispensable of these enzymes is pectinase, so named because it is specific to pectin. Pectin a polymer of repeating units of galacturonic acid is one of the essential structural molecules found in fruits and vegetables. Natural pectinases in the fruit hydrolyze it during ripening. In fruit chemistry, pectin is linked to methyl esters or other polymers. The number of methyl esters on a pectin backbone is described as the degree of esterification, or DE. A 1% solution of esterified pectin forms a strong gel at low pH in the presence of sucrose. Other polymers found with pectin might be composed sugars, such as repeating arabinose units (arabans) or chains of galactose (galactans). Pectins are frequently associated with cellulose and hemicellulose.
The tendency of pectin to form gels acts as both a blessing
and a curse for fruit processors. While adequate pectin is essential
to create gels in jams, jellies and fillings, the presence of this soluble
fibril in juice or juice concentrate is a problem for the production
of clarified juices. Every clarified-juice processor performs a pectin
test in which juice is mixed with several parts of alcohol until the
pectin precipitates into white threads. Once the precipitate is no longer
visible, the juice is deemed depectinized and can be concentrated or
bottled. Juices may not require complete depectinization if the final
product is a single-strength juice, but depectinization is essential
for the production of concentrated fruit syrups that are later reconstituted
into beverages. If a juice is not depectinized, colloidal pectin may
precipitate in the final beverage. Reconstituted juice may exhibit turbidity,
or a haze, if pectin remains in suspension, or it might combine into
larger particles that settle to the bottom of the bottle. Juice-concentrate
production is not possible without depectinization for other practical
reasons. Pectin contributes viscosity and causes problems for the operation
of the evaporator, the equipment that removes water and concentrates
the fruit sugars to a 70% sugar syrup.
The term pectinase refers to a mixture of specific enzymes that break down pectin by hydrolyzing the polymer backbone and its side chains. Pectin lyase, polygalacturase and methyl esterase are three types of enzymes that achieve the complete degradation of pectin. Pectin methylesterase does not reduce the length of the pectin backbone but strips off methyl esters. These side chains are important to the action of polygalacturonase, which cant bond to the backbone until the steric influence of the esters is removed. Once the methyl esters are removed, the pectin has available sites for the bonding of calcium ions. This crosslinking forms another type of gel. Unlike polygalcturonase, pectin lyase can hydrolyze methylated pectin.
Practice made perfect
Conventional wisdom dictated that the more types of pectinases added to the fruit pulp, the better, because the action of the enzyme mixture would more thoroughly macerate the pulp. This, in turn, would enable the expression of more juice in the press and would provide plenty of time to eliminate the pectin. The enzyme preparations used during the initial 30 or 40 years of fruit processing with enzymes were made by fungal organisms that produced mixtures of three pectinases, and sometimes they contained cellulase and hemicellulase activities as well. These additional activities acted synergistically to disassemble a wide variety of molecules of small and large molecular weights. Exposing the pulp to these enzyme mixtures released many soluble polymers into the juice, resulting in a product very high in soluble and insoluble solids. The solids and colloids were then filtered out to recover the clarified juice.
It became apparent during the 1990s that the use
and sometimes, overuse of these very effective hydrolytic enzymes
could cause problems as well as solve them. Fruit used for juice is
seldom of fresh, table-produce quality: it is undersized, sometimes
damaged, and frequently has higher ratios of skin and fiber to juicy
pulp. The goal of the processing industry is to push the envelope in
terms of extracting all the juice possible, reduce waste and make culled
fruit into a profitable business.
When enzymes were added liberally to macerate the fruit
(a process called liquefaction), they also liberally released other
degraded polymers and fibers, such as arabans. An unexpected haze developed
in the concentrates during storage, an emergency for juice processors
who sell on quality parameters that include clarity. Suddenly, the industry
was clamoring for the enzyme arabinase to eliminate the araban hazes
found in the juice. Pectinases are now sold with and without a specified
arabinase activity for processors who occasionally need to prevent the
formation of this haze. Araban haze is not common to all juice, but
has been observed in apple and pear processing.
As knowledge of specific enzyme activities and their actions
on fruit increased, conventional wisdom gave way to new technologies.
Fruit processors discovered that more enzymatic breakdown is not necessarily
more productive in terms of quality and yield of clarified juices. For
cloudy-juice processors, the overuse of enzymes can destabilize the
cloud and cause settling.
One advantage of new enzyme technology is the availability
of monocomponent enzymes of higher purity and strength. Gary Johnson,
regional marketing manager for the beverage business at Novozymes North
America, Inc., Franklinton, NC, explained how new products are addressing
this issue: For the fruit-juice processor who is just interested
in releasing juice without creating fine solids and colloids that clog
filters and impair pressing, we developed a pure pectin lyase that will
deliver the required juice yields without degrading the structure of
the macerated fruit required for efficient pressing. This pure pectin
lyase is known for its high activity at low pH and higher temperature,
making it an excellent choice for low-pH fruits. This enzyme is user-friendly
because it cannot cause problems if it is overdosed. It degrades only
the pectin and cannot harm the mash structure. Pectin lyase also cannot
create cellobiose, methanol or additional galacturonic acid, compounds
that are a concern to juice processors when using mixtures of pectinase
enzymes. There is no point in adding activities that are not required
when we can produce enzymes now that feature only the specific activity
required for optimal yield in the juice-recovery process.
Cellobiose, a dissacharide produced during cellulose degradation,
is an indigestible sugar that is associated with diarrhea in babies.
It is also considered to be an indicator of adulterated juice, since
cellulase enzymes could potentially break down some of the cellulose
in filter aids and become part of the pressed juice. Food companies
take the conservative position of limiting cellobiose in juice as a
safeguard, although scientists are still debating what levels are acceptable.
Pectin lyase is just as useful for producing cloud-stable juice as it is for clarified juice, since the presence of methyl esters is important to maintain the stability of the cloud and they are unaffected by pectin lyase. A typical enzyme dosage before the press ranges from 40 to 80 ml of enzyme per ton, with more required for fruits that have been in cold storage. Manufacturers add the enzyme before pressing, and again after pressing, until a negative alcohol test results. Since the amount of enzyme added is small compared to the amount of fruit-pulp treated, the enzyme is diluted first and metered in with a pump to disperse it throughout the mashed fruit before pressing the juice from the pulp. Pear, apple, grape and berry juices can be heated to 50º to 65º C to quickly depectinize them, since the higher the temperature, the faster the enzyme turnover. For premium juice concentrates with minimum browning and maximum aroma, juices are depectinzed at temperatures as low as 15º to 25º C. The lower temperature also inhibits the microbial counts from the native yeasts and bacteria that are found abundantly on these raw products.
Understand the enzymes
The selection of the enzyme best-suited to a particular fruit, problem or desired result is usually made by running a product test first on the bench-top and then in the processing plant. The results of these tests can be somewhat misleading, since fruit changes in composition from year to year in response to weather and harvest conditions, and among different varieties of the same fruit. If a plant processes several types of fruit, a different enzyme might be used depending on the fruits pH, the processing temperature, the ripeness or the plant personnels past experiences.
Living organisms produce enzymes that have unique characteristics
inherent in each production strain, even if the production organism
is the same species. That makes the job of enzyme selection somewhat
more complex than, say, buying a bag of citric acid. Actually, enzyme
use and selection requires a degree of skill that begins with understanding
the nature of the primary catalytic activities of the enzyme mixtures
and their pH and temperature optimums, and being able to recognize a
good and bad result when enzymes are used in the plant.
Ken Stewart, technical sales representative, GusmerCellulo Co., Fresno, CA, frequently consults with his customers in the Pacific Northwest, where the largest concentration of a wide variety of raw fruit is processed every year. My customers have a good understanding of the predominate activities in the enzyme products they use for fruit processing particularly how they affect the raw materials, he says. They know their substrate and the molecular basis by which it is hydrolyzed. It is useful to know how other processing aids may interact with enzymes. Bentonite, for example, will bind enzyme proteins and remove them from the juice. Inactivation of the enzyme is also important for the juice-concentrate industry, because reconstituted juice may be used in a variety of products that are later stabilized with gums or pectins that could be affected by residual enzyme activities. Customers who buy fruit concentrates may be checking for residual enzyme activity.
Checking in on citrus
Citrus is a rich source of pectin. After oranges are mechanically pulped, the raw juice is separated from the solid pulp residue. Water and pectinase are added to the pulp and additional juice is recovered during pulp washing. Some of the pulp is then added back to the juice extract. Pasteurization of the juice inactivates the native and the added pectinase enzyme extracts. Producers use high temperatures and vacuum evaporators to retain juice-concentrate flavor, and add pectinase before evaporation to prevent gelling. Manufacturers may also use it to extract essential oils from orange peel. The albedo portion of citrus peel consists primarily of pectin, so pectinase and cellulase enzyme blends were developed to facilitate the mechanical peeling of oranges and grapefruits. After the fruit is mechanically scored or pricked to allow exposure to the enzyme solution, the fruit is warmed and exposed to an enzyme bath to soften the peel. Mechanical peelers finish the job with a light cleanup by hand, resulting in a cleanly peeled final product. Dipping the peeled segments in citric acid, which denatures the enzyme and reduces microbial contamination, inactivates the enzyme.
Enzymes are also useful for processing whole fruit and vegetable pieces. Processed fruits can become soft and susceptible to handling damage, especially when cut into small-enough pieces for inclusion in fruit-flavored yogurts, baked goods or dessert toppings. A pure pectin methylesterase demethylates pectin on the surface of fruit or vegetable pieces. Once the enzyme cleaves the methyl ester and calcium is added, the pectin forms a calcium-pectin matrix that contributes to the firmness of cut produce after undergoing heating or freezing. This enzyme has been used to improve the firmness of strawberries, apples, cherries, mangoes, bananas, peaches, apricots, tomatoes and blueberries. The enzyme is added at 0.05% to 0.20%, based on the weight of fruit treated. Pectin methylesterase provides firming benefits in canned, individually quick-frozen, vacuum-infusion, high-pressure-sterilization and bulk-pack freezing processes. Unlike enzymes that are inactivated with freezing, it survives freezing and continues to provide a benefit in the final product.
Some enzymes with that wine
One of the most critical uses of enzymes in fruit processing is for winemaking. In white and red wines clarity is critical to final quality, as cloudy wines will never be in style. In winemaking, nearly every part of the process is crucial to quality, including the time and method of harvest, the transport of the grapes, the crushing, the pressing, and the hold times in the juice tank. Using a depectinizing enzyme is one of the simplest things winemakers can do to ensure final-product quality.
Winemakers use enzymes discriminately and conservatively.
Even during critical times such as the crush when
the grapes are loaded and trucks are lined up for miles to deliver their
sugary cargo waiting for juice to depectinize is not the time-limiting
factor it is in juice-concentrate production. During fermentation, the
juice is held in tanks for several days. Depectinization begins by the
action of endogenous enzymes in the grapes, so the winemaker can use
discretion in adding enzymes, depending on berry maturity and berry
variety, and adapt enzyme addition to his production parameters. Generally
pectinase is added early in the fermentation, before the alcohol content
becomes too high.
A more-valuable wine-production application for pectinase
is the addition of enzymes during pressing to extract additional color
and flavor from red grape skins. Wine flavor hinges on its tannins,
which are found in specific structures of the berry. Enzyme technology
has advanced sufficiently to selectively extract tannins known to contribute
to quality and flavor. Winemakers refer to these as soft tannins,
unlike the more undesirable tannins that are bound to protein and contribute
bitterness. The enzymes are fairly ineffective on the seeds a
fortunate benefit, since seed tannins should not predominate over the
tannins and polyphenols extracted from the pulp near the skins. The
skins and pulp cells also entrap the anthocyanins, and manufacturers
prefer fermenting red wines on the skins to extract as much color as
possible from skins before pressing. Using an enzyme allows more color
and flavor extraction from the berry cells during pressing.
But, as the winemaker knows, it is not how much color you have at the beginning: it is how much you have at the end of the fermentation that counts. Modern methods of analysis show that in wine, as enzyme levels increase, so does the quantity of stable anthocyanins, polyphenols, glucosides, tannin-flavans and flavonols. Typical use levels can be as low as 3 to 6 grams of enzyme preparation to 100 liters of must.
New solutions for protein problems
Protein content poses one of the standard obstacles when producing clarified wines. Tannin and protein can form colloidal precipitates that can settle out in the bottle as the wine ages and matures. Winemakers use various methods to remove protein; including heat and fining agents that pull proteins from the wine with positively charged molecules. Fining is done post-fermentation and before bottling. Most fining agents, such as bentonite, are not specific to protein, but also bind to some of the beneficial components that are found in clarified wines, including color and vitamins. Waste bentonite is also a disposal problem.
Another method of removing colloidal tannin-protein complexes
is cold stabilization. This method requires holding the wine until the
offending colloids eventually settle to the bottom. This requires time,
tankage and energy in a decade in which refrigeration and space represent
significant costs to the wine-growing regions of California, Oregon
and Washington. Cold stabilization is also used in grape-concentrate
processing to remove tartrates, tartaric-acid crystals that form in
grape juice during cold storage. Concord-grape juice is particularly
rich in tartrates that require removal before concentrating the juice.
Protein is also a problem in this process since it inhibits formation
of tartrate crystals, lengthening the stabilization period.
South Bend, IN, has developed a new protease that can hydrolyze heat-unstable
protein in wine and fruit juices. The use of this protease can replace
a substantial amount of bentonite for protein removal. Tom Wong, Ph.D.,
an advisor for Valley Research, sees many useful applications for protease
enzymes in wines and juices. Small peptides and amino acids from
the hydrolyzed protein could be utilized by the yeast and prevent stuck
fermentations the fermentations where the yeast quit before all
the sugar is depleted, he says. Protein in wine causes other
problems too, including foaming and inhibiting crystallization of potassium
bitartrate in cold stabilization of wine. Elimination of protein could
solve these problems also, resulting in more product recovery, and savings
in labor and energy costs.
Wong notes that in teaming up with the company, he has
been given the opportunity to demonstrate proteases for low-acid applications
that derive from the same organism that produces pectinase, A.
niger. This product offers new options for juice processors and
winemakers seeking alternative methods for eliminating protein reactions.
Proteases for winemaking were nonexistent for years because most of the proteases available for use in food systems do not exhibit activity at wine pH or perform very well in the presence of ethanol. FDA considers proteases GRAS for food use, and protease derived from A. niger is approved for winemaking in the United States, per Title 27 of the Code of Federal Regulations, vol. 1, for alcohol, tobacco and firearms.
Another use of enzymes in wineries involves a mechanism for flavor development that was reported in literature in the late 1980s. It has long been known that the action of the enzyme beta-glucosidase, an enzyme found in grapes and yeast, can improve white-wine aroma. This enzyme was found in varying amounts in pectinase enzyme extracts and, now that the benefits of this and similar enzymes activities are known, pectinases have been selected and marketed especially for this purpose. Beta-glucosidase is effective on glycosides chemical bonds linking important aroma compounds called monoterpenes. This enzyme is seldom used in red-wine production, since cleaving glycosides can destabilize the anthocyanin structure and compromise the wines color. The probability of success when using enzymes to release flavor depends on the grape varietal, the activity of the enzyme chosen and the initial quality of the wine to be improved. The enzyme is added after fermentation when sugar is low, since glucose is an end product and inhibits the enzymes action.
Like many aspects of winemaking, using enzymes for flavor
development is both art and science. Sensory evaluation is the preferred
method to determine the efficacy of the aroma development, and winemakers
will utilize products with a specified beta-glucosidase activity. For
white wines, especially muscatel or Gewürztraminer, cleaving these
aroma precursors has been shown to release the desired terpenols that
improve flavor. Other activities in pectinase reported to release aroma
are rhamnosidase, apiosidase and arabinofuranosidase, each specific
to respective chemical bonds. These precursors are also found in other
fruits and vegetables that contain similar types of compounds.
Another enzymatic flavor improver is an enzyme preparation
called naringinase. Oranges and other citrus fruits contain naringin,
a glycolated flavonone. This is the major bitter compound in citrus
and can be converted to less-bitter compounds by a similar mixture containing
rhamnosidase and a glucosidase. Wong explained that some pectinase enzymes
can eliminate off-flavors in wines as well, such as grassiness
or green characters of white wine, while also offering functional
benefits, such as better cold-settling of wine solids and more-compact
One reason that aromas are so much more detectable in
wines is the presence of ethanol, which volatilizes the aromas into
the nose so effectively. As flavor becomes more important in the production
of juice-based beverages, food companies will learn to use enzymes to
release endogenous fruit and vegetable flavors the way that flavor companies
and wineries do.
This and other new uses may be in the future for enzymes used in fruit or vegetable processing, and its likely that enzyme suppliers will offer more refinements to these valuable processing aids.
Suanne J. Klahorst is associate director of the California Institute of Food and Agricultural Research at UC-Davis, where federal grants fund the evaluation of enzymes for the conversion of food- and agricultural-processing waste to renewable fuels and bioactive chemicals.