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 “Reducing-Sugar Release Analysis” quantifies improved bioavail-able sugar content in commercial grower finisher diets after inoculation with BioPlus® YC

Background In modern livestock man-agement, feed additives play an important role in animal nutrition. They help im-prove the utilization of the nutrients in feed, leading to increased performance and profitability.

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.

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 Bacil-lus by determining the conversion of biomass (feed) with high fiber content into reducing-sugars that can be absorbed by the pig.

2. Illustrate 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,

Barletta, 2011). Exogenous enzymes can be used as catalysts to improve feed utilization, but a more efficient solution is needed.

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 indi-vidual 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.

Increased microbial fiber digestibility from Bacilli improves the availabil-ity 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 add-ing 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, how-ever, 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.

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.

Fig. 1 - Plant carbohydrate fractions Non-Starch-Polysaccharides (NSP) = NDSF+NDF. NDF = neutral detergent fiber, NDSF = neutral detergent-soluble fiber (includes all non-starch polysac-charides not present in NDF), NFC = non-NDF carbohydrates (Hall, 2003).

Plant carbohydrates

Cell contents

Organic acids Fructans

NDSF

Non-starch polysaccharides

NFC

NDF

ADF

Pectic substances

Galactans

ß-glucans

StarchesMono+oligo- saccharides

Hemicelluloses Cellulose

Cell wall

1.

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 glu-cose, 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 de-pendent 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 pro-cess: Reducing Sugars.

That is why we developed a unique in vitro feed assay to quantify the effect of inoculating feed with BioPlus® YC Bacillus strains on reducing-sugar content. It is also referred to as the Reducing-Sugar Release Analysis (Nielsen, 2014, 2013a+b).

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

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

3) Enables testing of individual feed samples for customers -> “Proof of concept”

The Reducing-Sugar Release Analysis in brief As illustrated in Figure 3, complete feed samples from individual pro-duction systems are collected and autoclaved at 121ºC for 15 min to kill other microorganisms. The feed sample(s) are then diluted with sodium

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 reduc-ing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. Gusakov et al. (2012) and Miller (1959).

100

95

90

85

80

75

70

65

60

P<

Crude Fat

.36

Crude Protein

.26

Crude Fiber

.02

Dry Matter

.05

Energy

.04

BioPlus® YCControl

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 aver-aged 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.

37ºC; 24hours; ph 6-6.5

Centrifugation Supernatant is used for

DNS + reducing sugar

DNS = 3.5dinitrosalicylic acid

3-amino-5-nitrosalicylic acid (absorbs light strongly at 540 nm)

Time 24 hours (T24)

Time 0 hours (T0)

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 centri-fuged and the supernatant is finally 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).

Proof of concept from adding BioPlus® YC to a grower finisher diet in the US The Reducing-Sugar Release Analysis was used to quantify the effect of inoculating a grower finisher diet with BioPlus® YC Bacillus strains on the reducing-sugar release from feed. As seen on Figure 4, the addition of Bio-Plus® 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.

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.

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 invest-ment required to add BioPlus® to 116,422.5 kg of feed equals 46.6 kg of BioPlus® YC.

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 Index Diet Composition

N in 290 290 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.

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.

T0 T24

BioPlus® YC 0.37±0.05 3.53±1.43

Control 0.41±0.18 1.29±0.77

Table 4 - Calculated Return-on-Investment in a commercial setting for produc-tion of 1,000 growers using date from Table 3.

Control BioPlus® YC Added value

Weight in, kg 16200 16200

Weight out, kg 68210 70350

Total gain, kg 52010 54150

FCR, g/g 117542.6 116422.5 -1120.1*

BioPlus® YC kg needed 46.569

Total kg pig produced for sale (total gain kg * live pigs)

50397.69

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.

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.

4

3.5

3

2.5

2

1.5

1

0.5

0T0 T24

BioPlus® YC

Control

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 • The unique (patent has been filed) Reducing-Sugar Release Analysis

from Chr. Hansen is now available for individual customers upon request.

• BioPlus® 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 us-ing 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, dkjaem@chr-hansen.com.

References Barletta, A. (2011). Introduction: Current Market and Expected Develop-ments. In “Enzymes in Farm Animal Nutrition”, CABI, UK, 2nd ed. 1-11.

Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determina-tion 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 carbo-hydrate 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) Ba-cillus 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 avail-able 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 polysac-charides degrading activities in pig feed by measuring changes in viscosity and reducing sugars. MSc Thesis, Aarhus University.

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