BioPlus® YC A Dual Strain Probiotic
Designed for Improved
Return on Investment
in Commercial Grower
Finisher Diets
By: Jack Egelund Madsen,
Strategic Product Manager
A novel “ReducingSugar Release
Analysis” quantifies
improved bioavailable sugar content in
commercial grower
finisher diets after
inoculation with
BioPlus® YC
Barletta, 2011). Exogenous enzymes can be used as catalysts to improve
feed utilization, but a more efficient solution is needed.
Background
In modern livestock management, feed additives play
an important role in animal
nutrition. They help improve the utilization of the
nutrients in feed, leading to
increased performance and
profitability.
Plant carbohydrates
Feed prices accounts for
about 70% of total pig production costs. Feed utilization, however, is not
optimal. Pigs cannot digest 15-25% of their feed rations because they
lack fiber or non-starch polysaccharide (NSP) degrading enzymes in their
intestinal tract (Table 1, Barletta, 2011). If NSP digestibility could be
improved it would significantly increase feed utilization, both directly and
indirectly. Increased fiber digestibility would also increase the availability
of other nutrients that are trapped within the fiber matrix. As a result,
the energy supply from the fiber itself would also increase, and fewer
nutrients will be lost to the environment.
Table 1 - Non-starch polysaccharides (NSP) in different feed sources (*)
Feed Ingredient
Amount of NSP
in feed, % of DM
NSP fraction contained in the feed
Cereals
10-15
Beta-glucans, hemicellulose (barley)
Corn
10-15
Hemicellulose + cellulose
Sorghum
10-15
N.D.
DDGS
25-35
Hemicellulose + cellulose
Wheat bran
25-35
N.D.
Soybean meal
20-35
Pectins
*Modified from: Nielsen et al., (Nielsen, 2014). N.D. not described.
The objective of this paper is to:
1. Introduce a novel high-through put method for quantifying the effect of
in vivo NSP enzyme production from BioPlus® YC dual strain Bacillus by determining the conversion of biomass (feed) with high fiber
content into reducing-sugars that can be absorbed by the pig.
2. I llustrate the potential return-on-investment from adding BioPlus® YC
dual strain Bacillus in grower diets in a commercial field trial.
How to unlock the nutritional potential of the NSP fraction
in feed
In modern pig production, feed utilization is not optimal. In fact, 15-25%
of feed rations are not digested because animals lack fiber or non-starch
polysaccharide (NSP) degrading enzymes in their intestine (Table 1,
In pigs which cannot produce NSP enzymes, supplementing their feed
with probiotic microorganisms, such as BioPlus YC, which can synthesize
NSP enzymes, has been shown to improve the nutritional value of individual feed ingredients and to stabilize the gut under commercial farm
conditions. The Bacilli would enable the degradation of the non-starch
polysaccharides (NSP) that cannot be digested by monogastrics alone,
and would utilize the 15-25% of the feed not being digested by pigs. See
Figure 1: Illustration of the NSP fraction from plant carbohydrates.
Cell contents
Cell wall
Pectic
Organic acids Mono+oligo- Starches Fructans
Hemicelluloses Cellulose
saccharides
substances
Galactans
ADF
ß-glucans
NDSF
NDF
Non-starch polysaccharides
NFC
Fig. 1 - Plant carbohydrate fractions
Non-Starch-Polysaccharides (NSP) = NDSF+NDF. NDF = neutral detergent
fiber, NDSF = neutral detergent-soluble fiber (includes all non-starch polysaccharides not present in NDF), NFC = non-NDF carbohydrates (Hall, 2003).
Increased microbial fiber digestibility from Bacilli improves the availability of other nutrients, including vitamins and minerals that are trapped
in the fiber matrix. Increased microbial fiber digestibility also increases
the energy supply from the fiber itself, and reduces the loss of nutrients to
the environment. This is illustrated in Figure 2, which shows the results
of a digestibility study on the effect of adding BioPlus® YC dual strain
Bacillus to a commercial grower finisher diet. The study found that adding BioPlus® YC to a grower finisher diet improves digestibility of: Crude
fat, protein, crude fiber, crude ash, dry matter and energy (2.2%-units
enhancement for protein, 1.1%-unit enhancement for energy). These
outcomes serve to improve animal performance and profitability.
The digestibility study provides valuable evidence supporting the benefit
of adding BioPlus® YC to feed in terms of utilization of nutrients and
energy in the feed. It is not feasible to conduct such a digestibility study
on an individual customer-specific basis, as it is both time-consuming and
expensive; for that, a high-through-put method is needed.
A novel “high-through-put” method for analyzing the nutritional
potential of the fiber fraction
NSP enzyme activity can be analyzed for each single enzyme (for example,
endo-cellulase or xylanase) that is produced by the BioPlus® YC dual
strain Bacillus (Skov, 2014). The breakdown of the NSP fraction, however, requires the action of multiple enzymes working in a coordinated
manner and is dependent on the specific feed source; information
regarding the quantity of individual enzymes is of less importance.
1.
Advantages of the Reducing-Sugar Release Analysis
1) The combined effect of all NSP enzymes produces by BioPlus® YC can
be tested in a high-though-put analysis
100
BioPlus® YC
Control
95
2) Provides easy comparison with either a control sample without
BioPlus® YC or to a specific standard curve for quantification of the
amount of total reducing sugars released
90
85
3) Enables testing of individual feed samples for customers ->
“Proof of concept”
80
75
The Reducing-Sugar Release Analysis in brief
As illustrated in Figure 3, complete feed samples from individual production systems are collected and autoclaved at 121ºC for 15 min to kill
other microorganisms. The feed sample(s) are then diluted with sodium
70
65
60
P<
Crude Fat
Crude Protein
Crude Fiber
Dry Matter
Energy
.36
.26
.02
.05
.04
Fig. 2 - Digestibility Study Conducted at a large Midwestern US swine
production company using BioPlus® YC
Titanium Dioxide was added to all diets 1 week prior to sampling. The pigs averaged 155 lbs bodyweight. Feed samples were collected from each feeder after
the pigs had consumed the diets for 4 days, either with or without BioPlus® YC.
Fecal samples were collected from 4 pigs in each pen on 2 consecutive days (d5
and 6) and fecal sample were pooled for analysis. Feed and Fecal samples were
analysis for: Crude Fat, Crude Protein, Crude Fiber, Crude Ash, Dry Matter,
Energy (Bomb Calorimeter). Digestibility Results: Adding BioPlus® YC to the
diets provided a numeric improvement in the digestibility of Crude Fat and Crude
Protein, and statistically significantly improvements in Crude Fiber, Dry Matter
and Energy digestibility. P value indicated on graph.
The key is to measure the degradation end-products of digested fiber:
reducing-sugars that can be utilized by the pig. A reducing-sugar is any
sugar that either has an aldehyde group or is capable of forming one to
allow the sugar to act as a reducing agent. Reducing sugars include glucose, glyceraldehyde and galactose as well as disaccharides, like lactose
and maltose.
The Bacillus subtilis and Bacillus licheniformis strains used in BioPlus
YC have been specifically selected for their ability to produce a wide
array of enzymes. The enzymes which are secreted by the bacillus are dependent on the substrate (feed source) that surrounds them. Since Bacilli
are most interested in their own survival, and they need to eat to survive,
they will produce the
enzymes that are most
advantageous to them.
This ultimately benefits
the pig as well. We can
measure this enzymatic
activity of the Bacilli by
simply measuring the
end result of this process: Reducing Sugars.
37ºC; 24
hours; ph 6-6.5
Time 0 hours (T0)
Time 24 hours (T24)
Centrifugation Supernatant is used for
®
That is why we developed a unique in vitro feed assay to quantify the
effect of inoculating feed with BioPlus® YC Bacillus strains on reducingsugar content. It is also referred to as the Reducing-Sugar Release
Analysis (Nielsen, 2014, 2013a+b).
DNS + reducing sugar
DNS = 3.5
dinitrosalicylic acid
3-amino-5-nitrosalicylic
acid (absorbs light
strongly at 540 nm)
Fig. 3 - The Reducing-Sugar Release Analysis
Commercial grower finisher feed are collected and autoclaved at 121ºC for 15 min
to kill potential microorganisms. The feed sample(s) are then diluted with sodium
phosphate buffer to ensure a pH=6-6.5 and split in two. One control sample and
one control sample plus BioPlus YC (1.28*106CFU /g feed). The samples are
incubated at 37ºC for 24 hours and CFU count is then re-analyzed. The samples
are then centrifuged and the supernatant is analyzed for the content of reducing
sugars using 3.5-dinitrosalicylic acid (DNS) as described by Gusakov et al. (2012).
DNS is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly
at 540 nm. Gusakov et al. (2012) and Miller (1959).
2.
phosphate buffer to ensure a pH=6-6.5. The feed sample is then split into
two samples: one control sample and one control sample plus BioPlus®
YC (1.28*106 CFU /g feed). The samples are incubated at 37º C for 24
hours and CFU count is then re-analyzed. The samples are then centrifuged and the supernatant is finally analyzed for the content of reducingsugars using 3.5-dinitrosalicylic acid (DNS) as described by Gusakov
et al. (2012). DNS is an aromatic compound that reacts with reducingsugars and other reducing-molecules to form 3-amino-5-nitrosalicylic
acid, which absorbs light strongly at 540 nm. Gusakov et al., (2012) and
Miller, (1959).
Proof of concept from adding BioPlus® YC to a grower finisher
diet in the US
4
The Reducing-Sugar Release
Control
Analysis was used to quantify
3.5
the effect of inoculating a
BioPlus® YC
grower finisher diet with
3
BioPlus® YC Bacillus strains
on the reducing-sugar
release from feed. As seen on
Figure 4, the addition of BioPlus® YC to the feed sample
resulted in a supply of more
nutrients to the animals in
the form of reducing-sugar
as compared with the control
sample (Figure 4, Table 2).
Feed samples with BioPlus®
YC deliver about 2-3 times
more reducing-sugars than
the control.
2.5
2
1.5
1
0.5
0
T0
Table 3 - Data from a commercial farm trial illustrating
The-Return-on-Investment using BioPlus® YC in grower diets.
As proof of concept a commercial farm trail was setup to investigate BioPlus® YC
in grower diets.*
Grower results
Control
BioPlus
N in
290
290
Index
Diet Composition
NE Mcal/kg
2.29
Weight in, kg
16.2
16.2
100
Crude Prot. %
16.9
Weight out, kg
68.21
70.35
103
Lysine, %
0.86
ADG, g/day
667
694
104
Methionine, %
0.57
FCR, g/g
2.26
2.15
95
Met. & Cryst, %
0.57
Mortality %
3.1
0.7
23
Phytase, IU/kg
500
*Commercial farm conditions (farm trial, 80159). Trial period: August-October
(78 days). Medium sanitary conditions. 1 feeding phase - Control: standard
grower diet and BioPlus®: Control diet + BioPlus® YC.
We can use the data from Table 3 to calculate the return on investment in
a commercial setting for the production of 1000 growers, as illustrated in
Table 4. It shows that the difference between adding BioPlus® YC to the
diet compared to a diet without BioPlus® YC (control) would be 1120.1
kg less feed in the production as a result of the improved FCR in the
BioPlus YC group (assuming the mortality is taking place at the final stage
in the production). The 1120.1 kg feed saved could be used to produce
521kg extra pig (FCR = 2.15). In addition, there will be an additional
3373.3 kg pig produced for sale as calculated by total gain kg* number of
pigs at the end of the period (997 and 969, respectively growers getting a
diet with BioPlus® YC or a control diet, Table 3 and Table 4). The investment required to add BioPlus® to 116,422.5 kg of feed equals 46.6 kg of
BioPlus® YC.
T24
Fig. 4: Reducing sugar units available for utilization by the pigs in feed
samples from an commercial US based grower finisher unit. Control samples
is feed without BioPlus® YC. N=4, BioPlus® YC ** significant. Data from Table 2.
Table 2 - Effect of adding BioPlus® YC on the release of reducing
sugar in commercial grower finisher feed (OD, mean ± st.dev) as
illustrated on Figure 4.
Table 4 - Calculated Return-on-Investment in a commercial setting for production of 1,000 growers using date from Table 3.
Control
BioPlus® YC
Weight in, kg
16200
16200
Weight out, kg
68210
70350
Total gain, kg
52010
54150
117542.6
T0
T24
FCR, g/g
BioPlus® YC
0.37±0.05
3.53±1.43
BioPlus® YC kg needed
116422.5
Control
0.41±0.18
1.29±0.77
Total kg pig produced for sale (total gain kg * live pigs)
Added value
-1120.1*
46.569
50397.69
Proof of return on investment: Commercial farm trial
To illustrate the benefit of BioPlus® YC´s in commercial settings a farm
trial was setup to study the effect of inoculating diets with BioPlus® YC
Bacillus strains on the performance in grower´s as a result of improved
reducing-sugar release from the feed and improved gut stabilization as
illustrated in Table 3. Increasing microbial fiber digestibility results in
higher bioavailability of reducing-sugars and other nutrients, vitamins
and minerals normally fixated by the fiber matrix, and that in turn leads
to a 4.8% improvement in FCR, 4.0% in ADG and a 2.4% unit reduction
in mortality.
53770.95
3373.26
The-Return-On-Investment from 1kg BioPlus® YC
kg less feed used
24.05
kg extra pig produced
72.43
* The 1120.1kg feed saved could be used to produce 521kg extra pig, assuming
a FCR at 2.15.
3.
Thus, the overall return on investment in the commercial farm trial can
be stated as follows:
Investment: 1 kg of BioPlus® YC will give you…
Return on Investment:
• 72.4 kg more pig
…on top of saving…
• 24 kg feed
Conclusion
•T
he unique (patent has been filed) Reducing-Sugar Release Analysis
from Chr. Hansen is now available for individual customers upon
request.
•B
ioPlus® YC (a dual strain Bacillus probiotic feed additive) from
Chr. Hansen delivers 2-3 times more reducing sugars, available for
absorption by the pig than the control.
• In the specific example shown above, the return on investment using BioPlus® YC in grower diets can be described as simple as: 1 kg of
BioPlus® YC results in a return on investment of 72.4 kg extra pig being
produced plus a savings of 24 kg of feed.
For more information regarding BioPlus® YC and the “Reducing-Sugar
Release Analysis” find us on World Pork Expo June 4-6, 2014, or contact
Strategic Product Manager Jack Egelund Madsen, [email protected].
References
Barletta, A. (2011). Introduction: Current Market and Expected Developments. In “Enzymes in Farm Animal Nutrition”, CABI, UK, 2nd ed. 1-11.
Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., vol. 31, 426-428.
Gusakov, A.V, Kondratyeva, E.G., Sinitsyn, A.P. (2011). Comparison of
two methods for assaying reducing sugars in the determination of carbohydrate activities. International Int. J. Anal. Chem. 1-4.
Hall, M.B. (2003). Challenges with non fiber carbohydrate methods. J.
Anim. Sci. Vol 81.
Nielsen, B., Nielsen, J., Styrishave, T., Cantor, M., Derkx, P. (2014) Bacillus producing NSP enzymes in vitro increase the amounts of available
sugars in feed. Spores Conference, RHUL, April 9-11, 2014
Nielsen, B., Nielsen, J., Styrishave, T., Cantor, M., Derkx, P., (2013a).
Bacillus producing NSP enzymes in vitro increased the amounts of
available sugars in feed. Proceedings of the Symposium on Gut Health
in Production of Food Animals, November 11-13th, 2013, Kansas City,
Missouri, p 18.
Nielsen, B., Nielsen, J., Styrishave, T., Cantor, M., Derkx, P. (2013b).
Bacillus producing NSP enzymes in vitro increased the amounts of available sugars in feed. Proceedings of the 6th European Spores Conference,
April, 9-11th, 2013, London.
Skov, W.J.M., (2014). Screening Bacillus strains for non-starch polysaccharides degrading activities in pig feed by measuring changes in viscosity
and reducing sugars. MSc Thesis, Aarhus University.
4.