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Nutritional and Metabolic Characteristics of Brassica Carinata Coproducts from Biofuel Processing in Dairy Cows Yajing Ban, Nazir Ahmad Khan, and Peiqiang Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02330 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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Journal of Agricultural and Food Chemistry

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Nutritional and Metabolic Characteristics of Brassica Carinata Coproducts from Biofuel

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Processing in Dairy Cows

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Yajing Ban1, Nazir Ahmad Khan1,2,*, and Peiqiang Yu1,**

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University of Saskatchewan, Saskatoon, SK, S7N 5A8, Canada

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2,

Department of Animal and Poultry Science, College of Agriculture and Bioresources,

Department of Animal Nutrition, The University of Agriculture Peshawar, 25130, KP, Pakistan

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*

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Tel: +1 306 966 4132; Fax: +1 306 966 4150

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E-mail: [email protected]

Corresponding author at manuscript submission stage:

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** Corresponding author

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Peiqiang Yu, Ph.D.

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Professor and Ministry of Agriculture Strategic Research Chair

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College of Agriculture and Bioresources, University of Saskatchewan

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6D10 Agriculture Building, 51 Campus Drive, Saskatoon

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Canada, S7N 5A8

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Tel:

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E-mail:[email protected]

+1 306 966 4132; Fax:

+1 306 966 4150

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ABSTRACT: The increased utilization of Brassica carinata in the biofuel industry in Canada

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has resulted in the large scale production of coproducts that can be potentially exploited as

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alternative protein ingredient in dairy ration. The objectives of this study were to investigate the

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nutritive value of carinata presscake and meal for dairy cows in terms of (1) nutrient and anti-

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nutrient composition; (2) rumen degradation kinetics of organic matter (OM), crude protein (CP)

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and neutral detergent fiber; (3) hourly effective degradation ratio and potential N to energy

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synchronization; (4) intestinal digestion of rumen undegraded protein (RUP); and (5) total

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metabolizable protein (MP) supply to the small intestine. Samples (n=3) of carinata meal,

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carinata presscake and canola meal (as reference feed), collected from three consecutive batches,

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were evaluated. Compared to canola meal, carinata presscake and meal had greater (P < 0.05)

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contents of CP (39.7% vs. 48.5%, 53.5% dry matter (DM)), with a high proportion of soluble

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crude protein (24.0% vs. 53.0%, 72.6% CP), resulting in their extensive degradation (59.2% vs.

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76.3, 89.3% CP) in the rumen. As a result, carinata presscake and meal supplied smaller (P
99%),

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sodium tungstate (Na2WO4·2H2O; purity 100%), boric acid (purity > 99.5%), sulphuric acid

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(purity 70%), ethyl ether (purity 99%), sodium borate (Na2B4O7·10H2O; purity 99.5%),

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hydrochloric acid (purity 38%), acetone (purity ≥ 99.5%), potassium phosphate (purity > 99%),

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sodium sulfite anhydrous (purity 98.4%), kjeltabs (0.15 g CuSO4 and 5.0 g K2SO4; N < 0.005%)

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and potassium hydroxide (purity 85%) were purchased from Fisher Scientific (Ottawa, ON,

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Canada). The enzymes, pepsin and pancreatin, were purchased from Sigma-Aldrich Co. (St.

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Louis, MO, USA). The Neutral detergent fiber solution containing 30 g sodium dodecyl

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sulphate, 18.61 g ethylenediamine-tetraacetic acid disodium salt, 6.81 g sodium borate, 4.56 g

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anhydrous sodium phosphate diabasic,10 ml triethylene glycol and heat-stable α-amylase (17400

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liquefon units/ml), and acid detergent fiber (ADF) solution containing 20 g cetyl trimethyl

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ammonium bromide in 1 N sulphuric acid were purchased from Ankom Technology (Macedon,

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NY, USA).

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Carinata Processing

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In this study carinata meal and carinata presscake were evaluated in comparison with

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commercial canola meal as a reference feed. The cold-pressing of carinata seeds from three

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consecutive batches was carried out in POS Bio-Sciences (Saskatoon, SK) according to the

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approved method of Agrisoma Biosciences Inc. (Saskatoon, SK). From each batch three samples

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of carinata presscake were collected, and no added heating was used during the cold-pressing.

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Similarly, three batches of carinata meal were produced from seeds received in three consecutive

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batches at Agrisoma Biosciences Inc. (Saskatoon, SK). For the meal production, mixed-colored

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carinata seeds (yellow and brown) were weighed, transferred into a 4 L plastic pail, and sprayed

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with water to maintain moisture content of 2.5%. After thorough mixing, the tempered seeds

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were flaked using the lab flaking mill and heated in beakers in a microwave oven (DMW904W,

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Findlay, OH, US), operated at a power of 900 W (1.33 W/g) and an irradiation frequency of

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2450 MHz for 2.5 min. Then all beakers were covered and transferred to a conventional oven for

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20 minutes at 95±3 °C. Later, the cooked flakes were pressed through a 5/16 die plate using the

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Gusta Laboratory Press (Gusta Manufacturing, Winnipeg, MB), and extracted oil during the

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crude-pressed process was collected. The laboratory Soxhlet extractor was then used to extract

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the residual oil using 6 liters of fresh hexane for 4 h. Lastly, the carinata meal was placed in a

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fume hood for a minimum of 36 h for air desolventizing. Three samples were collected from

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each batch of carinata meal. Samples from three batches of canola meal (n=3 from each batch)

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were provided by Federated Cooperatives Limited (Saskatoon, SK) as a reference.

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Chemical Analysis

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For chemical analysis the meals and presscake samples were ground (Retsch ZM-1, Brinkmann

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Instruments Ltd., Mississauga, ON, Canada) through a 1-mm screen, whereas for in situ

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incubation studies the samples were ground through a 2-mm screen. The contents of dry matter

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(DM; method 930.15), ash (method 942.05), crude fat (Cfat; method 954.02) and CP (method

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984.13) were analyzed according to the official methods of AOAC.14 The neutral detergent fiber

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(NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were analyzed according to

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the method of Van Soest et al.15 using the ANKOM A200 filter bag technique (ANKOM

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Technology Corp., Fairport, NY, US). Neutral detergent insoluble CP (NDICP) and acid

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detergent insoluble CP (ADICP) were analyzed according to Licitra et al.16 using the NDF and

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ADF residues. For determination of soluble CP (SCP) content, all samples were incubated in a

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borate-phosphate buffer for three hour, and filtered through #54 Whatman filter papers.16 Non-

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protein nitrogen (NPN) was estimated as the difference between total CP and precipitated true

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protein in tungstic acid.16 Total carbohydrate fraction (CHO) was calculated according to NRC 17

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as: CHO = 100 - (Cfat + CP + Ash). The contents of glucosinolates were determined by POS

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Bio-Sciences laboratories (Saskatoon, SK) according to the official method of the Canadian

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Grain Commission.18 The condensed tannins were determined using the HCl−butanol

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procedure.19

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Animals, Diets and Rumen Incubation

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Four rumen-cannulated (10 cm internal diameter; Bar Diamond Inc., Parma, ID, USA) non-

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lactating Holstein dairy cows (body weight, 680 ± 10 kg), were used for the in situ incubation

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trial. The procedures involved in the animal experiment was approved by the University of

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Saskatchewan Animal Research Ethics Board (Animal Use Protocol No. 19910012), and

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conducted according to the guidelines of the Canadian Council of Animal Care. 20 Cows were

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individually fed twice daily at 0800 and 1600 h with a total mixed ration (TMR) in a tie-stall

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barn at the Rayner Dairy Research and Teaching Facility (University of Saskatchewan,

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Saskatoon, SK), and given free access to water. The TMR was formulated with 56.5% barley

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silage, 12.2% hay and 31.3% concentrate according to the NRC requirements.17 The standard

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method of in situ rumen incubation as described by Khan et al.21 was used to determine the

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rumen degradation kinetics of the experimental feeds. The dried residues were weighed and

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pooled for chemical analyses as per treatment and incubation time within each run. The residues

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were ground by a coffee grinder (PC770, Loblaws Inc., Toronto, ON) for 10 s, repeated once, to

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obtain approximately 1 mm particle size. The residues were analyzed the content of CP and NDF

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as described earlier.

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Rumen Degradation Kinetics

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The first order kinetics degradation model with lag time was used to estimate the degradation

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features of OM, CP and NDF.22-24 The data was processed using the non-linear (NLIN)

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procedure of SAS 9.3 (SAS Institute, Inc., Cary, NC, US) and iterative least-squares regression

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(Gauss-Newton method). R = U + D × e 7


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Where R(t) is the residue (%) after t h incubation, U is undegradable fraction (%), D is

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degradable fraction (%), Kd is degradation rate (%/h), and T0 is lag time (h).

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The rumen undegraded fractions of OM (RUOM), CP (RUP) and NDF (RUNDF) were estimated using a passage rate (Kp) of 6%/h17, 24: RU% = U + D ×

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where RU is rumen undegradable fractions of OM, CP and NDF. The rumen degradable

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fractions were estimated as: % = + ×

+ !

"

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where ED is effective degradability of OM (EDOM), CP (RDP) and NDF (EDNDF), and S is

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soluble fraction (%).

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The hourly nutrient degradation was estimated based on the equations from Sinclair et al.

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Based on this study, the equation of hourly effective degradation ratios of N to OM was

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published by Nuez-Ortín and Yu.26

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Hourly ED (g/kg DM) = S + [D × Kd / (Kp + Kd)] × [1 − e−t × (Kd + Kp)],

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Hourly ED N/OMt = (HEDNt − HEDNt−1) / (HEDOMt − HEDOMt−1),

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where N/OMt is the ratio of N to OM at time t (g N/kg OM), HEDNt is effective

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degradability of N at time t (g/kg DM), HEDNt−1 is effective degradability of N at 1 h before

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time t (g/kg DM), HEDOMt is effective degradability of OM at time t (g/kg DM), and

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HEDOMt−1 is effective degradability of OM at 1 h before time t (g/kg DM).The optimum N to

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OM degradation ratio was considered to be 25 g N/kg OM to realize the most efficient microbial

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crude protein (MCP) synthesis. 26

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Intestinal Digestibility of Protein

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Digestibility of RUP in the small intestine was determined by three-step in vitro procedure of

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Calsamiglia and Stern.27 Briefly, 15 g of residues from 12 h in situ rumen incubation were

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weighed and mixed with 10 mL of 0.1 mol/L HCl solution (pH = 1.9) containing pepsin, and

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then incubated at 38 ºC for 1 h in a shaking water bath. After incubation, the solution was

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neutralized by the addition of 0.5 mL of 1 mol/L NaOH (pH = 7.8) and 13.5 mL of phosphate-

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buffer containing pancreatin. The mixture was subsequently vortexed, and incubated for 24 h at

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38 ºC in the shaking water bath, and vortexed every 8 h. At the end of 24 h incubation, 3 mL

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trichloroacetic acid (TCA) solution was added to the mixture in order to stop enzymatic activity

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and precipitate the undigested protein. Samples were then centrifuged for 15 min at 5000 rpm,

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and 5 ml of supernatant was analyzed for soluble N by the Kjeldahl method. The soluble protein

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was regarded as the digestible protein in the small intestine.

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Predicted Truly Absorbed Protein Supply and Feed Milk Value

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The metaboliable protein (MP) supplied to dairy cows was calculated as the sum of (1) truly

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absorbable rumen synthesized MCP in the small intestine (AMCP); (2) truly absorbable rumen-

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undegradable feed protein reaching the small intestine (ARUP); and (3) truly absorbable

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endogenous protein reaching the small intestine (AECP).17 The detailed description of the

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concepts, equations and formulas for estimation of AMCP, ARUP and AECP are given by Peng

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et al.28 The MP absorbed from the small intestine is partly used for milk production. The feed

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milk value (FMV) is the term used for estimating the efficiency of feed MP for milk

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production.29 The transfer efficiency of MP to milk is 0.67, and 1 kg milk contains

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approximately 33 g milk protein 17,29 Therefore, FMV can be estimated as:

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FMV = 0.67 ×

MPg/kg DM 33

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Statistical Analysis

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Data on the chemical profile, rumen degradation kinetics of nutrients, hourly effective

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degradation ratios, intestinal digestion of protein, MP supply to the small intestine and FMV of

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the carinata coproducts and canola meal were analyzed using the MIXED procedure of SAS 9.3

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(SAS Institute, Inc., Cary, NC, US). The model used for the analyses of nutrients and anti-

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nutrients profile was

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Yij = µ + Fi + eij,

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where Yij is an observation of the dependent variable ij, µ is the population mean for the

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variable, Fi is the effect of different coproducts as a fixed effect (different sources were treated as

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the replication), and eij is the random error associated with the observation ij. For the analysis of

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rumen degradation kinetics, hourly effective degradation ratios, intestinal digestion of protein

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and MP supply the following model was used,

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Yijk = µ + Fi + Sj + eijk,

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where Yijk is an observation of the dependent variable ijk, µ is the population mean for the

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variable, Fi is the effect of different coproducts as a fixed effect, Sj is the effect of different

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batches in the in situ trial as a random effect, and eijk is the random error associated with the

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observation ij. For all statistical analyses, significance was declared at P < 0.05, and trends at

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0.05 ≤ P ≤ 0.10.

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Results and discussion

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Chemical Profiles and Anti-nutritional Compounds of Carinata Coproducts 10



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The results of chemical profile, protein and carbohydrate sub-fraction compositions of B.

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carinata coproducts, meal and presscake, in comparison with canola meal are presented in Table

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1. There were no significant differences among carinata coproducts and canola meal for the

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contents of ash. Carinata presscake had a higher (P < 0.05) content (2.5% DM) of Cfat than

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carinata meal (0.3% DM). The presscake was produced after partial extraction of oil from

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carinata seeds by cold-pressing; whereas, from meal the residual oil was further removed by

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solvent-extraction process. This explains the higher content of Cfat in carinata presscake as

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compared to carinata meal. In agreement with our findings Theodoridou and Yu30 reported a

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higher content of Cfat in canola presscake as compared to canola meal. Notably, both coproducts

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of carinata had higher (P < 0.05) contents of CP than canola meal. A further comparison between

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the carinata coproducts revealed that the presscake had numerically lower content (48.5% vs.

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53.5% DM) of CP than the meal. This result is in accordance with the literature,31,32 and a

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plausible explanation is that the cold-press system of oil extraction results in lower oil recovery

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from the presscake compared to the solvent extraction, and as a result the presscake contains a

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higher content of oil and lower content of CP as compared to the meal.

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The CP subfraction composition revealed that compared to canola meal the SCP contents were

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more than two times higher (P < 0.05) in the carinata coproducts. The SCP is expected to

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degrade rapidly in the rumen, and may not be optimally utilized for MCP synthesis, that can

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lower the overall supply of MP to the dairy cow.33,34 Between the carinata coproducts, the

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presscake had higher (P < 0.05) SCP content (72.6% vs. 53.0% DM) than carinata meal. Mustafa

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et al.35 reported that compared to presscake the SCP content in solvent extracted meal is reduced

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by more than half after toasting, which they attributed to both high moisture and high heating,

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that prevailed during the solvent extraction process. Heating during solvent extraction can alter

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protein structures such as by the uncoiling of the pleated structures or by denaturing the protein

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molecules.34,36 The subsequent liberation of hydrophobic amino acids, racemization, and

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establishment of cross-linkages among the uncoiled peptides and between amino acids and

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reducing sugars can all reduce CP solubility. The contents of NDIP and ADIP were lower (P
0.10) in the Kd

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among the three coproducts. The greater (P < 0.05) D-fraction and smaller (P < 0.05) U-fraction

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was recorded for carinata presscake, while carinata and canola meal did not differ (P > 0.05) in

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these degradation characteristics. Among the three coproducts, carinata presscake had greater (P

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< 0.05) EDNDF (47.1% NDF), while carinata and canola meal did not differ (P > 0.05) in

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EDNDF. Our findings are consistent with literature values of McKinnon and Walker.31 However,

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another study indicated that presscake processing may not change rumen degradability of NDF.29

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Hourly Effective Rumen Degradation Ratios of Carinata Coproducts

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The optimal effective degradation ratio between N and energy supply to the rumen of dairy cows

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is 25g N/kg OM for achieving maximum MCP synthesis and minimum N loss to environment.

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lower degradation ratio indicates a shortage of N supply for MCP synthesis. Figure 1 shows the

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hourly effective N/OM degradation ratios of the three coproducts at different incubation times.

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The hourly effective N/OM degradation ratios of the two carinata coproducts were relatively

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higher than that of canola meal at the beginning (0 h) of incubation, with only the ratio of canola

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meal being less than the optimum (25g N/kg OM). Subsequently, a dramatic increase in the

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N/OM effective degradability ratio was observed between 0 to 2 h for the three coproducts, and a

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greater change (21 to 92g N/kg OM) was observed for canola meal. In the first 2 h the changes in

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N/OM degradation ratios of carinata meal and presscake were more or less similar and both

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attained a similar ratio (120g N/kg OM) after 2 h of incubation. However, compared to carinata

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meal, the N/OM degradation ratios of carinata presscake decreased rapidly from 120 g N/kg OM

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at 2 h to 14 g N/kg OM at 24 h, which is less than the optimum ratio (

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