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The newest old way to make enzymes

A subtle change has been occurring in the world's feed mills over the past decade. Enzymes are being increas-

ingly used in more rations. This is partially because nutritionists have a better under- standing of how enzymes work and because of the overwhelming evidence, have confi- dence that enzymes will improve the quali- ty of feed. Also, there are more feed-grade enzymes commercially available. Nutrition- ists have more options when they reach into their feed ingredient "tool box". Some feed manufacturers have likened the ability of enzymes to improve feed quality to what pellet mills did for feed quality in the 1950s.

The enzyme palette

Enzymes are biological catalysts, meaning that they accelerate the rate of naturally occurring biochemical reactions. For exam-

Table 1 - Enzyme activities of phytase produced through two different fermentation techniques Enzyme Submerged
Table
1
-
Enzyme activities of
phytase produced
through two
different
fermentation
techniques
Enzyme
Submerged
Solid-state
liquid
Phytase (PU/g)
1900
1900
-amylase
(FAU/g)
BDL
1
300
-gluconase
(BGU/g)
BDL
2700
Protease
(HUT/g)
BDL
9300
Xylanase
(XU/g)
BDL
500
Cellulase
(CMCU/g)
BDL
390
1 BDL=Below detectable
limits

The most common way to produce enzymes is through submerged microbial fermentation. However, by returning to a much older technology, solid-state fermentation, enzymes can be produced with a broader range of activities.

By Dr. Keith Filer

ple, phytic acid, the storage form of phos- phorus in seeds, will break down to its com- ponent phosphate molecules over time. However, enzymes speed up this process by several hundred-, or even several thousand- fold. The concept of using enzymes to produce different products—particularly in food—is not new. Scientists have known about the existence of enzymes for more than 100

years. The famous biochemist Louis Pasteur recognised that enzymes catalyse fermenta- tion reactions. In 1897, Eduard Buchner suc- cessfully isolated alcohol fermentation enzymes from yeast. (Actually, the word, "enzyme" means "from yeast"). In the late 1920s and early 1930s, research focused on the purification of a variety of enzymes including pepsin and urease. Today, more than 300 enzymes have been crystallised. Many have proved useful in food and feed production. Although feed manufacturers and live- stock producers use only a small fraction of the total amount of enzymes produced worldwide, they have come to rely on enzymes as very powerful tools for improving animal performance. Enzymes will become more valuable

as other additives, such as antibiotic growth promoters and pharmaceutical concentra- tions of zinc and copper, are banned. Most of the exogenous enzymes added to feed are hydrolases, meaning they break down feed particles into their component nutrients. Often, they compliment endoge- nous digestive enzymes that the animal's digestive tract already produces. For exam- ple, phytase enzyme increases the digestibil- ity of phytic acid in cereal grains and oilseed meals. This increases the amount of phosphate in the animal's intestinal tract that can be absorbed into the body. According to various studies, phytase can increase phosphate digestibility by 25-30%. The technology of feed-grade enzymes is constantly expanding. Less than 20 years ago, beta-glucanase and a few other non- starch polysaccharide-degrading enzymes were the only enzymes commercially avail- able to feed manufacturers. Now, a complete series of enzymes exists, including phytases to increase the bioavailability of inorganic phosphorus, galactosidases and proteases to free proteins trapped in the matrix of soy- bean meal and other oilseeds, lipases and xylansases to increase the metabolic energy content of feed.

Key features of SSF

Solid-state fermentation has several advantages when it comes to producing enzymes:

• Inexpensive, readily available substrates

• Applicable for non-genetically-modified (non-GM) microbes.

• Low start-up costs

• Short drying time

• Highly concentration of enzyme activity

• Heat and storage stable enzymes

• Side enzyme activity

Heat and storage stable enzymes • Side enzyme activity After inoculation, the koji is spread on

After inoculation, the koji is spread on sterilised trays and covered.

FEED MIX Volume 9 Number 2 2001

27

The proof is in the poultry

Dr. V. Ravindran of Massey University (New Zealand) recently evaluated the efficacy of a non-GM phytase enzyme produced through solid-state fermentation. The trial utilised 640 day-old broiler chicks and eight wheat-soybean experimental feeds. The feeds varied with respect to their available phosphorus content and supplemental non-GM, SSF phytase (Allzyme Phytase from Alltech). The experimental treatments were fed to the broilers for an entire 42-day growout cycle. The influence of the treatments on the performance over the 42-day trial period in male broilers is sum- marised in Table 2. Increasing the available phosphorus concentration improved weight gain and feed efficien- cy. The addition of phytase improved weight gain and feed efficiency. Available phosphorus and phytase sup- plementation had positive effects on toe ash. The bet- ter performance of birds fed phytase-supplemented diets compared to those fed diets supplemented with inorganic phosphorus may, in part, be attributed to side-enzyme activities present in the non-GM SSF phy- tase.

Table 2 - Effect of available phosphorus concentration and SSF phytase activity on performance and
Table 2 -
Effect of available phosphorus concentration and
SSF
phytase
activity on
performance and
toe ash
content of
male
broilers (0-42
days
of age)
Available
Added SSF
Gain 2
Feed
Feed:
Toe
ash 5
phosphorus
phytase 1
intake 3
gain 4
(%)
(%)
(Phytase units/kg)
(g)
(g)
0.30/0.20
-
2452
4626
1.911
9.68
0.36/0.26
-
2586
4844
1.873
10.57
0.42/0.32
-
2600
4869
1.880
10.67
0.48/0.38
-
2627
4741
1.832
10.69
0.30/0.20
500
2640
4590
1.775
10.70
0.30/0.20
1000
2684
4637
1.754
10.87
0.30/0.20
1500
2701
4584
1.730
10.82
0.30/0.20
2000
2781
4743
1.740
10.96
SEM
34.9
61.4
0.019
0.21
1 Allzyme
Phytase: One unit of phytase (PU)
is defined as
the
quantity of enzyme that
releases
one
micromol of
inorganic phosphorus per minute from 0.00015
mol/L
sodium phytate at
pH 5.5 at 37 ˚C.
2 Non-phytate P sequence
was used
days 1-21 and
22-42, respectively.
3 Phosphorus
effect
(linear, P<0.05).
Phytase
effect
(linear, P<0.001;
quadratic, P<0.001).
4 Phosphorus
effect
(quadratic, P<0.05).
5 Phosphorus
effect
(linear, P<0.05).
Phytase
effect
(linear, P<0.001;
quadratic, P<0.001).
6 Phosphorus
effect
(linear, P<0.01; quadratic, P<0.10). Phytase
effect
(linear, P<0.001; quadratic,
P<0.05).
effect (linear, P<0.001; quadratic, P<0.05). The covered trays containing the koji are loaded on racks

The covered trays containing the koji are loaded on racks and then placed in temperature and humidity controlled rooms to incubate for 5-7 days. The final material is dried, ground. standardised for enzyme activity, and packaged. The material can be extracted to produce a liquid phytase product.

Fermentation processes

Approximately 90% of all industrial enzymes—including those used in feed—are produced through submerged microbial fer- mentation. In this methodology, the microbes—usually fungi or bacteria that excrete large quantities of a specific enzyme—are submerged in nutrient-rich liq- uid media contained in a batch fermenter. The conditions inside the fermenter—pH, temperature, and oxygen—are tightly con- trolled to maximise microbial growth.

28 FEED MIX Volume 9 Number 2 2001

When the fermentation process is terminat- ed, enzyme-rich liquid media is dried, processed, and standardised. The attractive feature of this system is the high yields— especially when genetically-modified organ- isms are used. Solid state fermentation (SSF) is an alter- native to submerged microbial fermenta- tion for enzyme production. In a SSF system, microorganisms ferment a solid substrate. SSF is not a new concept, and its origins can be traced back to bread making

in ancient Egypt. The Japanese used a form of SSF to manufacture dietary protein from waste material. Modern examples of SSF are composting, silage production, cheese ripening, and mushroom cultivation. SSF has experienced wide use in Asia in the pro- duction of certain foods and beverages, such as sake and soy sauce. However, until recent- ly, enzyme suppliers have overlooked this unique technology as a viable production system. Establishing a SSF system begins with the screening, selection, isolation, and propaga- tion of a microbial culture that produces large amounts—in some cases more than 400 times the normal amount—of the desired enzyme. These cultures are used to inoculate the SSF system. Production of the microbes used to inoculate the system is done on a small scale. Sometimes a tradi- tional submerged fermentation system is used to generate the culture. Bacteria, yeast, and fungi that can grow on solid substrates are viable candidates. However, filamentous fungi, such as Aspergillus niger, have the greatest advan- tages in SSF systems that produce enzymes for feed applications. The next step is the preparation of the substrate. SSF systems can utilise minimally processed byproducts—such as wheat or rice bran—and other inexpensive, readily avail- able material, such as soyflour as the sub- strate. Often the ratio between protein and carbohydrate sources is a determining fac- tor in enzyme yield. The substrate must be heat and pressure sterilised to destroy any infective microbes—

particularly yeast—and their spores. The moisture content of the substrate is adjust- ed to 45-50%. The low free moisture content of the substrate is one of the most impor- tant features of SSF. Most of the water is bound to the substrate. This maximises the exposure of the microbes to air, which stim- ulates microbial activity and—more impor- tantly—enzyme production. Also, the low moisture reduces drying time and energy costs during downstream processing. The selected microbial culture is seeded onto the sterilised substrate and mixed to create a material called "koji" - a Japanese word. Usually 500 ml of starter culture can inoculate 1 ton of substrate. The koji is loaded into a reactor and allowed to fer- ment. There are three basic types of reac- tors— tray, packed-bed, and agitated. The tray reactor is the most widely used. It consists of a series of 35 x 20 cm sterilised stainless steel trays. A thin layer—5-7 cm—of koji is applied to the trays. The trays are cov- ered, placed on a stainless steel rack, and housed in a controlled environment (98% humidity) room. Workers follow very strict aseptic practices to avoid contaminating the system. To produce feed-grade phytase, the fermentation process usually takes 5-7 days. After the fermentation is complete, the material is dried, ground, standardised, and packaged. Alternatively, the fermented material can be extracted to produce a liq- uid enzyme product. A facility with 10,000

Where do enzymes come from?

Currently, the global market for enzymes is approximately US$1 billion per year. This figure is expect- ed to grow by 10% within the next five years. In livestock feeds, enzymes enhance digestibility and nutrient utilisation and destroy anti-nutritional factors. However, the use of enzymes in livestock and poultry feeds is small. Even when pet food is considered, feed use accounts for only about 1% of the total. Most of the industrial enzymes—about 45%—are proteases and lipases used in detergents. Food processing - mainly amylases, pectinases, and isomerases - comprise another 45% of the total.

trays can make a sufficient quantity of enzyme in one year to supplement 6-8 mil- lion tons of feed. Packed-bed and agitated reactors are being developed to increase enzyme produc- tion capacity. However, over-heating, aera- tion, and condensation inside the vessel are challenges that are being addressed. Also, in deep bed systems, certain layers could become deficient in oxygen.

Superior enzyme quality

Evidence is mounting that enzymes pro- duced through SSF are qualitatively differ- ent from those made by submerged microbe fermentation. SSF systems that use non-GM microbes often produce high activities of the desired enzyme as well as lower but sub- stantial activities of other enzymes—also known as "side activities." For example, an SSF-phytase product had measurable pro-

tease and beta-glucanase activities (Table 1). In another case, an SSF-produced glucosi- dase was found to be more heat-tolerant than one produced in submerged microbial fermentation. A microbial feed-grade phy- tase produced with SSF technology had greater stability during storage than one produced through traditional fermentation. When tested on three different feed ingre- dients and a complete feed, SSF protease had a significantly greater protein digestion capacity than the same type of enzyme pro- duced through submerged microbial fer- mentation. The future of the SSF will include new and better enzymes. In addition, it may be adapted to produce other feed ingredients, such as flavours, functional carbohydrates, and microbial by-products. Clearly, this technology will form the basis for improved feed quality.

FEED MIX Volume 9 Number 2 2001

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