Ruminal Biohydrogenation Kinetics of Defatted Flaxseed and

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Ruminal biohydrogenation kinetics of defatted flaxseed and sunflower is affected by heat treatment Saman Lashkari, Lone Hymøller, and Soren Krogh Jensen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03008 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 11, 2017

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Journal of Agricultural and Food Chemistry

Ruminal biohydrogenation kinetics of defatted flaxseed and sunflower is affected by heat treatment Saman Lashkari*, Lone Hymøller and Søren Krogh Jensen Author address:

Department of Animal Science, Aarhus University, AU Foulum, DK-8830 Tjele, Denmark. *Corresponding Author: Saman Lashkari, Department of Animal Science, Aarhus University, AU Foulum, DK-8830 Tjele, Denmark. Tel: +45 50245737, Fax: +458715 6076 Email: [email protected]

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ABSTRACT: The effect of heat treatment on biohydrogenation of linoleic acid (LA) and

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linolenic acid (LNA) and formation of stearic acid (SA), cis-9, trans-11 conjugated LA (CLA),

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trans-10, cis-12 CLA and trans vaccenic acid (VA) was studied in in vitro incubations with

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diluted rumen fluid as inoculum and partly defatted flaxseed (DF) and partly defatted sunflower

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(DS) as test feeds. Feeds were heated in a laboratory oven at 110 °C for 0 (unheated), 45 or 90

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min. Michaelis−Menten kinetics was applied for quantifying biohydrogenation rate. The DF

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heated for 90 min showed the lowest biohydrogenation rate of LNA and LA, indicated by the

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lowest Vmax value (P < 0.04 and P < 0.03, respectively). The DS heated for 45 min had the

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lowest biohydrogenation rate of LNA, indicated by the lowest Vmax value (P < 0.04). In

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conclusion, heat treatment decreased biohydrogenation of LA and LNA in DF and LNA in DS.

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KEYWORDS: rumen biohydrogenation, linoleic and linolenic acid, heat treatment.


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INTRODUCTION

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Dairy products, such as milk, contribute to the intake of essential nutrients in human

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populations.1 However, consumption of milk has declined over the last decades, as nutritional

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guidelines have advised limiting the consumption of saturated fatty acids, which to a significant

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proportion originate from dairy products and milk.2 Oilseeds and vegetable oils are the main

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sources of unsaturated lipids in the ruminant diets. Oilseeds are preferred because of less adverse

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side-effects on rumen fermentation.2,3 Recently, there has been a renewed interest in using

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oilseeds in animal rations as they can improve the fatty acid profile of milk and dairy products.3

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Supplementation of dairy cow diets with oilseeds, a rich source of polyunsaturated fatty acids

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such as LNA and LA, is the most common approach of producing polyunsaturated fatty acid-

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enriched dairy products.3,4 Sunflower and flaxseed are good candidates, as both are rich in

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polyunsaturated fatty acids, with sunflower being a source of LA (66% of the total fatty acids),

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while flaxseed is rich in LNA (56% of the total fatty acids).4 However, achieving an increase in

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the concentration of polyunsaturated fatty acids in dairy products is challenging because most

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unsaturated fatty acids are extensively biohydrogenated in the rumen.5 The ruminal

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biohydrogenation process is responsible for chemically altering dietary unsaturated fatty acids in

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the rumen, decreasing the proportion of unsaturated fatty acids, but also forming several other

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unsaturated fatty acids, including CLA and trans C18:1. Some intermediates of ruminal

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biohydrogenation, including VA (C18:1 trans-11) and CLA isomers, provide functional health

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benefits for the consumer.6

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The concept of protection of unsaturated fatty acids against ruminal biohydrogenation has

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received major attention in the past decade.4 Various chemical and physical processes have been

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proposed for protecting unsaturated fatty acids of oilseeds against ruminal biohydrogenation.7



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Heat processing is the most commonly used physical method for changing the biohydrogenation

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rate of oilseeds.8 Application of heat to oilseeds is a well-documented way to increase rumen by-

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pass of protein and the resulting denaturation of the protein matrix surrounding the fat droplets

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may also protect unsaturated fatty acids against ruminal biohydrogenation.9

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Although the absorbed fatty acid isomers affect animal performance and milk quality, the

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quantitative flow of individual isomers is not predictable. Estimating ruminal biohydrogenation

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rates of different processed oilseed is an important prerequisite in order to quantify relations

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between dietary fatty acids and the composition of fatty acids appearing in dairy products. We

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hypothesized that heat treatment will reduce the biohydrogenation rate of LNA and LA and

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change appearance rate of CLA and other intermediate biohydrogenated fatty acids.

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MATERIALS AND METHOD

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Defatted flaxseed and sunflower preparation and heat treatments. Flaxseeds and de-

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hulled sunflower seeds were purchased from a commercial feed company. Flaxseeds and

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sunflower seeds were first grinded on a Moulinex coffee mill (Moulinex S.A., Paris, France) and

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subsequently milled through a 1 mm screen on a Retsch ZM200 centrifugal mill (Retsch GmbH,

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Haan, Germany). Flaxseed and sunflower contained 94.3% and 93.7% dry matter (DM),

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respectively. To reduce the fat content, the oilseed were defatted twice with diethyl ether (VWR,

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Radnor, Pennsylvania USA ) to 16.8% and 23.2% residual fat for defatted sunflower and

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flaxseed, respectively. Defatted seeds were heated in a laboratory oven at 100 °C for 45, and 90

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min in heat resistant air tight plastic bags (ITS film & paper products, Apeldoorn, The

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Netherlands). A sample of unheated defatted flaxseed and sunflower was kept as control

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treatments. Buffer soluble nitrogen was determined according to the method described in

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NorFor.10


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Donor Cows. The study was carried out at Aarhus University, Department of Animal

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Sciences, Denmark, and animals were kept in accordance with the Danish Ministry of Justice

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Law No. 1306 (November 23, 2007) on animal experiments and care of experimental animals.

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Three standard heifers fitted with ruminal cannulas were used for donation of rumen fluid.

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Animals were fed a diet of 4.0 kg/d oven dried hay, 2.0 kg/d barley straw, and 2.8 kg/d of

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concentrate consisting of: barley (40%), soybean meal (10%), rapeseed meal (3%), sugar beet

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molasses (3%), and a vitamin and mineral mix (4%).

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In vitro incubation procedure. The in vitro incubation procedure was performed as

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previously described by Petersen and Jensen.11 Rumen fluid was filtered through a single layer of

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cheesecloth and immediately transferred to the laboratory in sealed preheated vacuum thermos

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flasks. Eighteen milliliters of rumen fluid was transferred to 50.0 mL test tubes from Greiner Bio

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One International GmbH. Prior to the transfer of the rumen fluid, 0.5 g of DF or DS and 18.0 mL

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of buffer solution were added to the tubes. The buffer was prepared according to the method of

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McDougall.12 The lid of the test tube was punctured with an injection needle and fitted with a

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syringe to allow gas to escape from the test tubes without compromising the anaerobic

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environment of the test tube. Incubations were stopped at 0, 1, 2, 3, 4, 8, 12, 16, 24 and 30 h by

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placing test tubes containing buffered rumen fluid and seeds in ice slurry. Subsequently samples

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were stored at -20 °C, freeze-dried and stored at -20 °C until further analysis. Throughout the

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experiment pH was monitored.

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Fatty acid analysis. Lipids were extracted in a mixture of chloroform and methanol

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according to the method of Bligh and Dyer13 after acidification by boiling in 3.0 mol/L HCl for

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1.0 h, using modifications and recommendations published by Jensen.14 Briefly, 150 mg freeze

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dried sample was weighed into a culture tube and extracted with 3.0 mL chloroform, 4.0 mL



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methanol and 1.5 mL distilled water and added 5 mg of internal standard C17:0 (heptadecanoic

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acid, Sigma Aldrich, St. Louis, MO). Lipids were quantified as fatty acid methyl esters according

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to a two-step methylation process of an acid-catalyzed method followed by a base-catalyzed

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method. The extract was acidified with 1.0 mL of methanolic HCl at 80 °C for 10 min, and fatty

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acid methyl esters extracted with 2.0 mL of pentane. After evaporation of the pentane, fatty acids

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were methylated with 2.0 mL of NaOCH3/methanol (0.5 N) for 10 min at 50 °C. Fatty acid

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methyl esters were subsequently extracted with 1.0 mL of heptane and analyzed on a gas

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chromatograph (Hewlett-Packard 6890 series, Agilent Technologies, Palo Alto, CA, USA)

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equipped with an automatic column injector (Hewlet Packard 7673), a capillary column of 30 m

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× 0.32 mm ID, 0.25 µm thickness (Omegawax 320; Supelco, Sigma- Aldrich), and a flame

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ionization detector. The initial temperature was set to 86 °C and raised to 200 °C at a rate of 2

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°C/min. The 200 °C temperature was held for 5 min and finally raised to 220 °C at a rate 5

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°C/min. Fatty acids were identified by comparison of retention times with external standards

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(GLC-68C, Nu- Prep-Check, Elysian, MN, USA).

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Kinetic Calculations. According to the Michaelis−Menten equation: V = Vmax ×

[s] [Km] + [s]

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a Hanes plot is a straight line. The results determined in the in vitro biohydrogenation experiment

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were analyzed by linear regression according to the Hanes plot as the time ([s], h) against the

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time divided by the amount of ALA or LA disappeared or SA and intermediate fatty acid formed

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(h/g/kg DM). Vmax (g FA/kg DM) was calculated as the reciprocal slope, and KM (h) was

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calculated as the intercept multiplied by Vmax and is the time for 50% reduction in ALA and LA

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concentration or 50% increase in SA and intermediate fatty acid formation. For ALA and LA

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their initial concentrations were subtracted from the concentration at the time when the 6 ACS Paragon Plus Environment

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incubation was stopped (e.g., time = 0 h subtracted from time = 0, 1, 2, 3, 4, 8, 12, 16, 24 and 30

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h), giving the total sum of products from the biohydrogenation at a given time. Rumen

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biohydrogenation is a complex system, where formation of the different intermediates can

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originate from isomerization, desaturation or hydroxylation along the different biohydrogenation

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pathways,11 therefore modeling of intermediates is difficult. In the current study modeling of

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intermediate fatty acids was attempted under the assumption that the concentration would

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increase over time as a result of biohydrogenation. The results are to be considered as the

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apparent appearance of intermediate fatty acid for the reasons stated above.

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Statistical analyses. Experimental data were analysed in a completely randomised design

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using the general linear method procedure of SAS.15 The effect of cow and interaction between

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cow and treatment was tested, but no significance was found, and so cow was included as a

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random effect in the final model. Heat processing was considered the only source of variation

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and the statistical model was: Yij = µ + Ti + eij

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where µ is the over-all mean, T is the fixed effect of heat treatment duration i (0, 45, 90 min) and

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eij is the random residual error. The dependent variable Yij represents the response for the ith

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duration of the heat treatment. Random effects were assumed normally distributed with mean

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value 0 and constant variance e ~ N (0,σ2). Means were compared using Tukey’s multiple range

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tests.

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RESULTS

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Chemical composition and nitrogen solubility of seeds. There is no significant difference in

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dry matter, fat and crude protein content between unheated and heat treated DF and DS (Table

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1). The highest nitrogen solubility was observed in unheated DF (P < 0.01). Also, protein



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solubility of unheated DS and DS heated for 90 min were highest and lowest, respectively (P

Ruminal Biohydrogenation Kinetics of Defatted Flaxseed and

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