Effects of Heat Treatment of Green Protein on In situ protein

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Effects of Heat Treatment of Green Protein on In situ protein disappearance and In vitro Fatty Acid Biohydrogenation Mohammad Rashed Chowdhury, Saman Lashkari, Søren Krogh Jensen, Morten Ambye Jensen, and Martin Riis Weisbjerg J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02176 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

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Journal of Agricultural and Food Chemistry

Effects of Heat Treatment of Green Protein on In situ protein disappearance and In vitro Fatty Acid Biohydrogenation

Mohammad Rashed Chowdhury1,2, Saman Lashkari1*, Søren Krogh Jensen1, Morten AmbyeJensen3, Martin Riis Weisbjerg1

Authors addresses: 1

Department of Animal Science, Aarhus University, AU Foulum, Blichers Alle 20, Post Box 50,

DK-8830, Tjele, Denmark. 2

Present address: Department of Biochemistry and Chemistry, Faculty of Biotechnology and

Genetic Engineering, Sylhet Agricultural University, Bangladesh. 3

Department of Engineering, Aarhus University, Hangøvej 2, 8200 Aarhus N, Denmark.

*Corresponding Author: Tel: +45 50245737, Fax: +45 8715 6076, Email: [email protected]

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ABSTRACT: Soluble protein extracted from leaves and stems of grasses and forage legumes is

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defined as green protein. The present study was conducted to evaluate in situ green protein

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degradability, intestinal protein disappearance and in vitro fatty acids biohydrogenation (BH) in

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dairy cows. Three green protein concentrates (red clover, ryegrass, and grass clover) were heat

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treated as follows: oven-drying at 70 °C, subsequent autoclaving at 121 °C for 45 min and for

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grass clover also spin flash-drying. Freeze-dried green protein was considered as a control

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(untreated). Autoclaving and oven drying of green protein reduced the crude protein (CP) and dry

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matter (DM) degradability. The linolenic acid (LNA) BH rate was lowest in heat-treated grass

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clover concentrate (P < 0.01). In conclusion, green proteins are heat sensitive, and oven-drying

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can be an appropriate method to increase the amount of protein and unsaturated fatty acids

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escaping from the rumen.

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KEYWORDS: in situ, in vitro, degradability, biohydrogenation, green protein


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INTRODUCTION

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Green protein concentrates originate from the extraction of various green biomass like grasses

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and legumes. Composition and value of green protein are comparable to other protein-rich

16

feedstuffs and are suitable for both ruminant and non-ruminant animals.1, 2 High yielding dairy

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cows require high-quality rumen undegradable protein supplement to meet their protein

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requirements for milk production.3 Supply of slowly degradable rumen feed protein has a special

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value for young growing ruminants, and for lactating dairy cows in early lactation.4 Dairy cows

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fed with legumes such as red and white clover silages have increased concentrations of

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polyunsaturated fatty acids (PUFA) in their milk.5 The concept of increasing the beneficial fatty

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acids (FA) content in dairy products for human consumption, including conjugated linoleic acid

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(CLA) isomers and vaccenic acid (trans-11, C18:1) has received a great deal of attention in the

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past decade.6, 7

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Heating is one of the most common methods to decrease the degradability of crude protein in

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the rumen and increase the nitrogen (N) efficiency use in dairy production,8 and it also reduces

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the in situ ruminal disappearance of FA of feed ingredients such as soybean, sunflower oil and

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seed, lupin seed and barley.9, 10 However, the application of heat should be in balance to avoid

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overprotection of feedstuffs. Different feed processing methods such as oven heating, roasting,

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extruding and autoclaving have been used to protect feed proteins from ruminal degradation.11 In

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numerous studies of heat treatment, autoclaving or moist heating with low temperature more

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effectively reduced crude protein degradability than dry heating.10, 12, 13 In addition, application of

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heat treatment may also protect PUFA against microbial BH and therefore rise the concentration

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of PUFA in meat and dairy products.14 Several studies have reported positive effects of heat

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treatment on protein degradability and FA disappearance of feedstuffs in cereals/grain or seeds. 3 ACS Paragon Plus Environment


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However, in vivo data on protein degradability and FA biohydrogenation (BH) of the green

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protein obtained from red clover, ryegrass and grass clover in dairy cows are scarce. To our best

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knowledge, the concept of utilisation of green protein in dairy cows is novel, and degradability of

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heat-treated green protein in the rumen of dairy cows has not been examined before. Thus, the

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present study was conducted to study the possibilities to manipulate the rumen degradability and

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BH of fatty acids in green protein using heat treatment.

42 43

MATERIALS AND METHODS

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Sample Preparation and Heat Treatment. Three green protein concentrate samples (red

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clover, ryegrass and grass clover) were used in the present experiment. The red clover and

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ryegrass pastes were collected from the pilot plant at Aarhus University, AU Foulum, Denmark,

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with input capacity of 1000 kg/h wet biomass. The processing included maceration in an

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industrial shredder (UNTHA RS 50), wet fractionation by screw press (Vincent, CP-10), protein

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precipitation at pH 4 obtained by natural lactic acid fermentation at 38 ᵒC followed by

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centrifugation in a decanter centrifuge (Alfa Laval, Foodec 219).

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The grass clover concentrate was sampled from a large scale production with 10000 kg/h wet

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biomass. The main difference in the processing of the biomass was introduction of a more

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controlled lactic acid fermentation process by inoculation of Lactobacillus salivarius BS 1001, in

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order to precipitate the protein from the green juice at its isoelectric pKa (pH 4). The resulting

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wet protein concentrate, with a dry matter w/w% of 30%, were stored at -20 ºC. A part of the wet

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protein concentrate from the demonstration test was dried in an industrial spin flash dryer and

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used directly in the present study. The drying was carried out at KMC A.M.B.A., Denmark (DK-

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7470 Karup), using their existing facilities for drying potato fiber. After defrosting, 4 ACS Paragon Plus Environment

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approximately 1 kg of each paste was dried at 70 °C in a forced air oven, and 0.5 kg paste was

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freeze-dried (Scanvac Coolsafe type 55-4). Half of all the oven-dried samples were subsequently

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heat-treated for 45 min at 121°C in an autoclave (steam pressure 0.3 Mpa) by using heat resistant

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plastic bags. An additional 0.5 kg spin flash-dried grass clover protein originating from the

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demonstration test as described above was obtained. All oven-dried, autoclaved, freeze-dried and

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spin flash-dried samples were milled through a 1.5 mm screen for in situ, a 0.5 mm screen for in

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vitro BH and a 1.0 mm screen prior to chemical analysis.

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Animals. The current study complied with the guidelines of the Danish Ministry of Justice

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(Act no. 726, 1993) with respect to animal experimentation and care of animals used for

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scientific studies.

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Three rumen fistulated (#1C, Bar Diamond Inc., Parma, ID, USA) Danish Holstein Friesian

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dry cows were used for in vitro rumen incubations. Cows were fed twice daily at 08:30 h and

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17:30 h at maintenance level. The complete ration (DM basis) consisted of 2 kg barley straw, 4

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kg artificially grass dried hay, 2.8 kg concentrate and 200 g minerals daily and 2.15 g vitamins

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daily. The concentrate mixture consisted of soybean meal 100 g/kg, barley 400 g/kg, oat 400

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g/kg, rapeseed meal 30 g/kg, sugar beet molasses 30 g/kg and minerals 40 g/kg. The forage to

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concentrate ratio of diets was 68:32 throughout the experimental period.

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Two lactating multiparous Danish Holstein cows fitted with T-shaped duodenal and ileal

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cannula were used to determine total tract digestibility. Both cows were fed with 60:40 forage

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(grass and maize silage) to concentrate ratio (DM basis) during the mobile bag incubations. The

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diets consisted of a total mixed ration (TMR, g/kg DM); grass silage 380, maize silage 390,

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rolled barley 98.2, rapeseed meal 48.6, soybean meal 40.5, calcium 0.7 and vitamin-mineral 5 ACS Paragon Plus Environment


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premix 3.0. All cows had free access to fresh drinking water. The average milk yield recorded for

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both cows was 37±7 kg/day.

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In Situ Study. Rumen in situ protein degradation was determined using nylon bag techniques

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according to the standard NorFor procedure.15 Briefly, air-dry Dacron bags (7×6 cm, 38 µm pore

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size) filled with the approximately 1±0.01 g ground (1.5 mm) sample and 12 mg/cm2 sample to

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surface area ratio as suggested by Nocek.16 The bags were incubated in the rumen for 0, 2, 4, 8,

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24, 48 and 96 h in each of the three dry cows. After rumen incubation, all bags were rinsed in

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running cold water and subsequently washed using a domestic washing machine for 10 minutes

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using 2 × 22 L water (25°C). Residues were transferred to (N) free filter paper (retention value 2,

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Whatman AGF 607-90 mm) for DM and CP analysis.

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Water Soluble Fraction. The water-soluble fractions of green protein samples for both DM

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and CP were measured as described by Weisbjerg et al.16 The milled sample (0.5 g) was weighed

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out in triplicate and soaked in 40 mL of water for 1 h. Samples were filtered with N free filter

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paper and washed with 4 × 40 mL of demineralised water. True water solubility was determined

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as the disappearance.

99 100

Total Tract Disappearance. Total tract disappearance was determined using the mobile bag

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technique according to Hvelplund and Weisbjerg.17 The bags (6×6 cm, 12 µm pore size) were

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sealed after loading 1.0±0.01 g and ruminally pre-incubated in three dry cows for 16 h, each

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sample replicated twice in each cow (six observations for each treatment). To simulate abomasal

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digestion, all bags were incubated in HCl (pH = 2.4) for 1 h and subsequently incubated for 2 h in 6 ACS Paragon Plus Environment

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pepsin-HCl solution (200 mg pepsin dissolved in 2 L HCl) at 40 °C in Daisy incubator

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(ANKOM, Macedon NY, US) with rotation. Thereafter, bags were inserted into the small

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intestine of two lactating cows through the T-shaped duodenal cannula. All the bags were

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collected from faeces. After faecal recovery, bags were machine-washed, and residues were

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transferred to the N-free filter paper using the same procedure as for rumen bags to determine the

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total tract DM and CP disappearance. Total tract indigestible CP was estimated as the CP residue

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in the mobile bags.

112 113 114 115

In Vitro Study Samples Used. For the in vitro study, all grass clover (oven-dried, autoclaved, freeze-dried and spin flash-dried) samples were used to assess the BH of unsaturated long chain fatty acids.

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Preparation of Rumen Fluid and Buffer Solution. Before the morning feeding, rumen fluid

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was collected from the same three dry cows in the same way as in the nylon bag study. Rumen

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content was collected by hand through the rumen cannula, and the fluid was obtained by straining

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rumen contents through double-layered cheesecloth. The fluid was immediately transferred to

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sealed and preheated vacuum flasks under anaerobic conditions and transported to the laboratory.

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The buffer solution was prepared according to the method of McDougall.18

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In vitro Incubation. In vitro incubation performed in triplicate with the three strained rumen

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fluids (from each of the three cows) according to Petersen and Jensen.19 In vitro incubation was

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run by rumen fluids of each cows and three cows made up the triplicate per treatment. Briefly,

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500 mg of ground (0.5 mm) grass clover concentrate, 18.0 mL strained rumen fluid and 18.0 mL 7 ACS Paragon Plus Environment


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McDougall buffer solution were transferred to 50 mL CELLSTAR® tubes (Greiner Bio-one

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GmbH, Kremsmunster, Austria). The lid of the tube was punctured with an injection needle (0.8

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× 25 mm), and then equipped with a syringe (20 mL; Becton, Dickinson and Co, Franklin Lakes,

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USA) to allow gas escape from the tubes without compromising the anaerobic environment. The

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tubes containing buffered rumen fluid and feed materials were put in a water bath at 38 °C for 0,

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1, 2, 3, 4, 8, 12, 16, 24 and 30 h. After incubation, test tubes were placed on ice slurry to stop

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fermentation and were frozen. The complete samples (a total of 120 observations) were freeze-

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dried and stored at -20 °C until further FA analysis.

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Chemical Analysis. Dry matter of feed and residue samples was determined by oven-drying

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at 103 °C for 18-20 h. 20 Ash content was determined by combustion at 525 °C for 6 h in a muffle

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furnace (Method 923.03).21 Total N content was determined by the Kjeldahl method (AOAC,

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1990) and CP as N×6.2521. Amino acids of feed samples were determined by following the

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method described by Tayyab et al.22, which is based on oxidation with performic acid and

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subsequent hydrolysis with HCl, followed by quantitative determination of individual AA using a

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Biochrom B20 automated AA analyser. Some AA such as valine, isoleucine and serine were

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corrected with a factor of 1.06 as they were prone to oxidation to a moderate degree of hydrolysis

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by the addition of hydrochloric acid.23

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Fatty acids were analysed by extracting lipids in a mixture of methanol and chloroform.24 FA

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were converted into methyl esters according to Jensen.25 Briefly, 450 mg of freeze-dried samples

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were weighed out in culture tubes and after acidification in 3.0 mol/L of HCl for 1 h at 80 °C,

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lipids were extracted with 3.0 mL chloroform, 3.0 mL methanol, 1.5 mL distilled water and 5.00

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mg of internal standard C17:0 (hectadecanoic acid, Sigma-Alrich, St. Louis, MO). The extracts

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were centrifuged for 10 min at 2000 g. Afterward, 1.0 mL of extract was transferred to new 8 ACS Paragon Plus Environment

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culture tube (WHEATON; 16×100 MM; culture C-tubes with cap round bottom, USA),

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evaporated to dryness under a N2 stream and then methylated according to Petersen and Jensen.19

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In this procedure, extracted samples were methylated with 0.8 mL NaOH (2%) in methanol,

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airtight with argon gas (Ar) and placed in an oven for 15 min at 100 °C. After cooling, 1.0 mL of

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boron trifluoride reagent was added with Ar and placed at 100 °C for 45 min. Finally, FA methyl

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esters were extracted with 2.0 mL heptane and 4.0 mL saturated NaCl solution followed by

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centrifugation for 10 min at 2000 g. A gas chromatograph (Hewlet Packard 6890, Agilent

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Technologies, Palo Alto, CA, USA) equipped with a capillary column of 30 m × 0.32 mm i.d.,

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0.25 µm thickness (Omegawax 320; Supelco, Sigma-Aldrich), an automatic column injector

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(Hewlet Packard 7673) and a flame ionization detector were used for quantifying the FA as FA

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methyl esters. The primary temperature was set at 170 °C and the temperature was increased at a

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rate of 2 °C/min to 200 °C, held for 5 min and finally raised to 220 °C at a rate of 5 °C/min.

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Peaks were identified by comparison of retention times with external standards (GLC 68C, Nu-

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Prep-Check, Elysian, MN, USA) for long-chain FA.

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Calculation. Since no lag phase was observed for disappearance of DM and CP, the DM and

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CP disappearances were fitted using the PROC NLIN in SAS (9.4 version, SAS Institute Inc.) to

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the equation Deg (t) = a + b (1– e–ct) without considering the lag time. The effective rumen

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degradability (ED) was calculated according to Ørskov and McDonald26 using a 0.05 h-1 rumen

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fractional passage rate as ED = a + b (c/c + k) where Deg is the percentage disappearance at time

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t, t is incubation time (hours), a is soluble fraction, b is degradable fraction but not soluble, c is

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the degradation rate of fractional b (% h-1) and k is the fractional passage rate (0.05 h-1). The

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soluble fraction, potentially degradable fraction and ED subsequently corrected for loss of 9 ACS Paragon Plus Environment


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particles for both DM and CP. Particle loss was calculated as the difference between 0 h

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disappearance and true water solubility as reported by Weisbjerg et al.27

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Total tract digestibility of DM and CP was calculated as the disappearance from mobile bags.

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Intestinal digestibility of rumen pre-incubated CP was then calculated on the basis of effective

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ruminal degradability (ED) and total tract digestibility as described by Hvelplund et al.28

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The disappearance rate of LNA and LA and the appearance rate of vaccenic acid (VA) and

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stearic acid (SA) were estimated using the non-linear model29. The data of LNA and LA from

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each in vitro run were fitted using the PROC NLIN in SAS (9.4 version, SAS Institute Inc.) to the

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equation Qt = exp (1-exp-(k (time- L)) where Qt is the amount (g/kg DM) of fatty acid at time t (h), k the

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disappearance rate of LA and LNA (h) and appearance rate of VA and SA (h), L the lag time (h)

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and t is the incubation time (h).

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Statistical Analysis. All statistical analyses were performed with R version 3.4.0 (R core

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team, 2017). Differences in chemical and AA compositions between the treatments (except for

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spin flash dried) were analysed using the linear model function lm with treatment as fixed effects.

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Differences in degradation parameters, SID and total tract disappearance were analysed for the

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effect of treatment and the interaction between the treatment (oven and autoclave) and grass

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species by using the lmer function from the lme4 package30 with treatment and species as fixed

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effects and cow as a random effect. The effects of heat treatment on grass clover were analysed

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using the same model with heat treatment as fixed effect and cow as a random effect. The results

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are stated as least square mean and standard error of the mean (SEM) for each treatment.

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Statistical significance was determined by P ≤ 0.05 and regarded as tendencies by P ≤ 0.10.

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RESULTS 10 ACS Paragon Plus Environment

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Chemical Composition. The different heat treatments did not affect the chemical composition

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of green proteins (Table 1). The ash concentration was the highest in red clover concentrate (284

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g/kg DM). The buffer-soluble CP (SCP) and the ratio between SCP and CP (SCP: CP) were not

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affected by heat treatment (P > 0.05) while the heat treatment numerically reduced the SCP in red

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clover and grass clover concentrate.

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Amino Acid Composition The AA composition of heat-treated green protein is shown in

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Table 2. Total AA concentration was affected by heat treatment (P = 0.01), and the highest and

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lowest total AA concentrations were observed in freeze-dried grass clover concentrate (291 g/kg

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DM) and autoclaved ryegrass concentrate (238 g/kg DM), respectively. The level of some

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individual AA, especially arginine, cysteine, histidine, lysine and methionine was affected by

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heat treatment (P < 0.001) and lysine content was the lowest in outocalved heat-treated green

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protein.

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In Situ Rumen Degradability. In situ degradation characteristics for DM and CP are

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presented in Table 3 and Table 4, respectively. The soluble fraction (a) of CP in grass clover

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concentrate significantly increased by the heat treatment (P = 0.01) and the highest soluble

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fraction was observed for oven-dried grass clover concentrate (130 g/kg CP) (Table 4). For the

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degradation rate (c) of potentially degradable fraction (b) of CP, there was an interaction between

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treatments (oven and autoclave) and grass species (P = 0.03). Freeze-dried grass clover

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concentrate had the highest c value of CP (18.9% h-1). The ED of CP was affected by the

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interaction between the heat treatment and grass species (P < 0.0001). The highest ED was

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recorded for freeze-dried grass clover concentrate (740 g/kg CP) and the lowest for autoclaved 11 ACS Paragon Plus Environment


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red clover concentrate (105 g/kg CP). The a fraction of DM significantly varied for all heat-

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treated grass clover concentrate (P = 0.003) and the highest a fraction of DM was found for

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autoclaved grass clover concentrate (198 g/kg DM), while the lowest was found for autoclaved

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red clover concentrate (68 g/kg DM). The interaction between the treatment and grass species

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was significant (P ≤ 0.05) for both the b fraction and the degradation rate of b fraction (Table 3).

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Heat treatment decreased the b fraction and c values of DM. The highest c value was found for

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spin flash-dried grass clover concentrate (12.5% h-1). The ED for DM was affected by interaction

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between treatments and grass species (P < 0.0001). The highest ED for DM was found for freeze-

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dried grass clover concentrate.

229 230

Total Tract Disappearance. Total tract DM and CP disappearance values varied between the

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treatments and grass species (P < 0.001, Table 5). The lowest values were found for all

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autoclaved samples (P < 0.0001). Freeze-dried grass clover concentrate showed a higher total

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tract disappearance (approximately 94% CP) than other samples (Table 4). The RUP value was

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highest for autoclaved red clover concentrate (895 g/kg CP). An interaction was found between

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heat treatment and grass specie for the SID of RUP (P < 0.01). The highest SID was found for

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oven-dried red clover concentrate (87% RUP), while lowest was found for autoclaved red clover

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concentrate (7% RUP).

238 239

In Vitro Biohydrogenation. The BH kinetic parameters of LA and LNA are presented in

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Table 6. Figure 1 displays the changes in the FA amount during the in vitro incubation for oven-

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dried grass clover concentrate. The heat treatments significantly affected the disappearance rate

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of LNA (Table 6), while no significant difference was observed for the disappearance rate of LA 12 ACS Paragon Plus Environment

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(Table 6). The freeze-dried grass clover concentrate had the highest disappearance rate of LNA

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(P < 0.04). No significant differences were observed for the lag phase in LA and LNA.

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Estimation of appearance rate CLA isomers (cis-9, trans-11 and trans-10, cis-12 CLA) with the

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first order kinetic model was not possible due to a small CLA pool size and the lack of CLA

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accumulation in fermentation flasks. However, the BH pattern of cis-9, trans-11 CLA and trans-

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10, cis-12 CLA was greatly affected by heat treatments as shown in Figures 2 and 3, respectively.

249 250

DISCUSSION

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Chemical Compositions. In the present study, heat treatment did not affect the nutrient

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composition (Table 1). The heat treatment numerically reduced the SCP concentration in heat-

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treated red clover concentrate and grass clover concentrate as reported by Goelema et al.31 In

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agreement with our results in another study, protein solubility in oven-dried forage silage

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decreased compared to freeze-dried.32 Reduced SCP decreases the rumen degradation of heated

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feedstuffs and therefore increases the RUP values.33 This might be due to increased levels of

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intermediately (B2) and slowly (B3) degradable CP fractions that are gradually increased upon

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heating.34 Although no significant difference was observed in SCP between the treatments,

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autoclaving numerically reduced the SCP of red clover concentrate and grass clover concentrate

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by 53% and 24%, respectively, compared to freeze-dried samples. Mustafa et al.10 reported that

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moist heating at 127 °C for 30 min decreased the SCP concentration of sunflower seed by 83%

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and increased the neutral detergent insoluble CP by 131% compared to unheated sunflower seed

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without any negative effect on acid detergent insoluble CP. In agreement with our findings, it has

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been reported that both dry or wet heat processing decrease CP solubility.13,

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extensive heat treatment damaged the protein quality by increasing the concentration of acid 13 ACS Paragon Plus Environment

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In addition,


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detergent insoluble CP that was reported for canola36, 37 and soybean meal.38

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Amino Acid Compositions. In the present study, extensive heat treatment decreased the

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content of total AA due to a drastic reduction of some individual AA in this experiment. The

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autoclave treatment of green protein decreased the lysine content from 21 to 14% compared to

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freeze-dried samples. A similar effect was found in rapeseed cake where an increase in

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temperature to 142 °C decreased the lysine content of rapeseed cake.39 Lysine is one of the most

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heat-labile AA and is often damged at levels 5 to 15 times higher than the other AA.40,

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addition, excessive heating may reduce the concentrations of other AA along with lysine like

275

aspartate, arginine, histidine, cysteine and methionine. This result might be due to the formation

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of Maillard products caused by reducing carbohydrates or maybe the direct formation of cross-

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links between AA during heating which makes such AA more resistant to acid hydrolysis during

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analysis.41, 42 However, moderate heat increases the total essential AA flow to the duodenum and

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can increase the AA absorption.43 In contrast, excessive heat treatment not only reduce the lysine

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content of feedstuffs but also the proportion of lysine which is available.41

41

In

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Rumen Degradability. Heat treatment generally increased the rumen escape fraction of CP

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and reduced the fractional rate of degradation for grass clover concentrate compared to freeze-

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dried samples (Table 4). In agreement with our results, Arieli et al.44 reported a lower rate of

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degradation in expanded barley, wheat and maize. Lund et al.39 reported that the degradation rate

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at 115 °C was most pronounced for grains and less for protein feeds (rapeseed cake, peas and

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guar meal). In our present findings, there was a tendency (P = 0.06) that heat treatment decreased

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the potentially degradable fraction of green protein CP. However, a decreased potentially 14 ACS Paragon Plus Environment

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degradable fraction for heated green protein was not expected. It seemed that for all autoclaved

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samples, the degradation curve became more linear compared to the exponential curve for other

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treated samples as also found by Lund et al.39 and Dakowski et al.41 This indicates that the

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degradation rate for autoclaved samples was very low at the start of the incubation period. Doiron

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et al.35 also found a similar decreasing trend of rumen degradable fractions for Vimy flaxseeds

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(74.2% to 64.5% CP) when the duration of autoclaving at 120 °C increased from 20 to 60 min.

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Moist heat markedly reduced effective protein degradability in the current study by 79%, 53%

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and 45% for red clover concentrate, ryegrass concentrate and grass clover concentrate,

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respectively, compared to oven-dried samples. Similar results have already been reported in

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cereal44 and protein meal.39 Extraction of other cell content as e.g sugars together accompanied

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by protein extraction might be an explanation for high sensitivity to the moist heat. However,

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different responses to heat treatment in red clover concentrate, ryegrass concentrate and grass

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clover concentrate can possibly be attributed to different sensitivity of different feedstuffs to

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moist heat. Therefore, the susceptibility to heat treatment of the examined green proteins varied

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widely, demonostrating that extrapolations from one green protein species to another species may

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be misleading.

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The DM degradation in the rumen usually followed the same pattern as seen for CP, indicating

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that the majority of the change in fractions of DM due to treatments is changes in the CP

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fraction22,

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treatment (P < 0.0001), and it was also found to be the highest for freeze-dried samples followed

309

by spin flash-dried grass clover concentrate. In agreement with our results, Lopez et al.32 reported

310

that DM degradability in freeze-dried ground forages was higher than the oven-dried. This

311

differences can be explained as a higher rapidly degradable fraction in freeze-dried samples

35

. Similar to CP, the effective ruminal degradability of DM was affected by the



312

Page 16 of 36

compared to oven-dried and autoclaved samples.32, 45

313 314

Total Tract Disappearance. The total tract disappearance was affected (P < 0.0001) by

315

different heat treatments (Table 5). Dakowski et al.41 observed that total tract disappearance of

316

rapeseed meals heated to 140 and 150 °C was severely decreased. The RUP values were higher

317

(P < 0.0001) for autoclaved samples than for the other heat treatments. Other researchers also

318

reported higher RUP values for autoclaved protein sources than for unheated sources.35, 37, 38, 46

319

McKinnon et al.36 stated that a RUP value may be compromised if post-ruminal digestion is

320

reduced by overheating.

321

The calculated amount of CP disappearing in the small intestine out of total CP intake (DEP)

322

varied between treatments in all green protein species (P < 0.004). The results of total tract

323

disappearance of CP showed that post-ruminal disappearance of CP in oven-dried and spin flash-

324

dried grass clover concentrate was higher than that of autoclaved grass clover, indicating that low

325

ruminal CP disappearance was compensated by intestinal digestion. This finding agreed with the

326

hypothesis of Hvelplund et al.28 showing that the intestine has a capacity which is more than

327

sufficient for CP digestion. This result is in line with a previous study47 which reported that the

328

feeds with low ruminal disappearance of CP had high post-ruminal disappearance because of the

329

compensatory digestion in the small intestine, however, the amount of compensatory digestion in

330

the small intestine is depended on the protein source and type of processing. Higher SID in oven-

331

dried samples also suggests that only primary Maillard products were formed, indicating that no

332

intestinally indigestible terminal Maillard products was formed.48 However, it seemed that

333

autoclaving had an adverse effect on SID of green protein. Probably the temperature and duration

334

of the treatments were sufficient to damage the green proteins. In agreement with our findings, 16 ACS Paragon Plus Environment

Page 17 of 36


335

McKinnon et al.36 concluded that heating canola meal to a temperature of 145 °C reduced the

336

ruminal and total tract disappearance of the DM and CP fractions. This fact might be due to the

337

formation of indigestible Maillard products during heating.49 In addition, the reduced lysine

338

content in the autoclaved green proteins (Table 2) is in line with lower SID, and the results may

339

prove the formation of Maillard products that are indigestible in the intestine. It is assumed that

340

there may be a negative correlation between the intestinal or total tract digestibility of N and acid

341

detergent insoluble N.36, 37, 50 Our findings suggest that the SID of all the autoclaved green protein

342

concentrates was adversely affected by the outoclaved treatment. Furthermore, due to the reduced

343

ruminal degradability, autoclave treatment increased the RUP values but subsequently decreased

344

the intestinal disappearance of undegraded CP. However, oven-dried samples showed a higher

345

DEP than other heat treatment. In addition, the low SID in autoclaved green protein showed that

346

green proteins are more valnurable to heat damge than the other protein source.13

347 348

In Vitro Biohydrogenation of Fatty Acids. Heat treatment is one of the most commonly used

349

physical approaches used in feed processing that can protect PUFA from ruminal BH.51 The

350

lower disappearance rate of LNA in heat-treated grass clover concentrate could correlate with the

351

lower protein solubility. The lower protein solubility in heat-treated grass clover concentrate may

352

be due to protein denaturation which probably make proteins more resistant to microbial

353

degradation, and may decrease the rate of LNA BH. The prolonged lag phase may confirm the

354

lower releasing rate of LA and LNA. In addition, the lower disappearance rate in LNA and the

355

high numerically lag phase for both LNA and LA in heat-treated grass clover concentrate are in

356

line with the lower protein degradation rate (Table 4), and it seems that the protein denaturation

357

may protect some PUFA from BH. Heat treatment may protect protein against degradation by 17 ACS Paragon Plus Environment


Page 18 of 36

358

denaturing the protein matrix, and it has been reported that fat droplets are protected by the

359

protein matrix.51 Therefore, we speculated that the reduced protein degradation rate and ruminal

360

protein degradability may cause a lower releasing rate of LNA and a prolonged lag phase for both

361

LNA and LA disappearances in heat-treated grass clover concentrate. In agreement with our

362

results, Boufaied et al.52 found that fatty acids might be protected physically by the denatured

363

protein matrix, decreasing the rate of release and thus decreasing BH of PUFA. However, the

364

disappearance rate of LA was not affected by heat treatments, and it seems that response to heat

365

treatment depends on type of PUFA. In agreement with our results, Lashkari et al.53 reported the

366

different responses of LA vs LNA in heat-treated defatted flaxseed and sunflower. The different

367

responses of LA vs LNA most likely due to a partly different location of LA and LNA in the

368

plant. The most of the LNA is located in the chloroplast membrane, whereas LA may be located

369

in the cell membranes. Thereby LNA is likely partly more closely associated to the Rubisco

370

proteins in the chloroplast and more likely to be trapped when the surrounding protein is

371

denaturated.

372

The lag phase is supposed to be the time needed for lipolysis and/or for a proliferation of

373

rumen microorganisms to have a sufficient number of microorganisms to lipolyse and

374

hydrogenate PUFA. Although the lag phase for the LA biohydrogenation rate did not differ

375

significantly, the lag phase increased 100, 230 and 297% in oven-dried, autoclaved and spin

376

flash-dried, compared to freeze-dried grass clover concentrate, respectively. In addition, the lag

377

phase for LNA increased 42, 109 and 225% in oven-dried, autoclaved and spin flash-dried,

378

compared to the freeze-dried grass clover concentrate, respectively. Despite the same

379

disappearance rate in LA, the high lag phase observed with the heat-treated may protect the LA

380

against BH. These results also showed that heat treatment, which decreased the disappearance 18 ACS Paragon Plus Environment

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381

rate and increased the lag phase in LNA, could be a practical approach to bypass and protect

382

LNA against the microbial BH and thus rise the concentration of LNA in dairy products.14

383

The highest lag phase for VA appearance was observed in spin flash-dried grass clover

384

concentrate. These results showed the same trend as for the lag phase in SA formations in spin

385

flash-dried grass clover concentrate. In addition, the results demonstrated that the lag phase

386

observed for formation of VA and SA followed the same trend as LNA and LA disappearances.

387

The highest lag phase in LNA and LA in spin flash-dried grass clover concentrate is in line with

388

the low amounts of CLA isomers during the incubation, and that could be due to low isomerase

389

activity. Although the reason for a high lag phase in VA formation in spin flash-dried grass

390

clover concentrate still is not clarified, changing the microbial ecosystem and/or inhibiting the

391

reductase activity of ruminal microorganisms may have been responsible for the lag phase in VA

392

formation. However, the heat-treated grass clover concentrate with the same LNA disappearance

393

rate and different lag phases showed that the delay in disappearance of LNA and LA might be

394

reversible, either by an adaptation of rumen microbes, which would produce more enzyme, or by

395

cell proliferation.54

396

Appearance of CLA isomers during incubations of LA and LNA with rumen fluid is known to

397

be transient and much less influenced by end products and other intermediates.55 Figures 2 and 3

398

indicated the high variation of CLA isomer content during the incubation and obviously showed a

399

transitory pathway for CLA isomer formation. The results of the present study showed that

400

formation of cis-9, trans-11 CLA and trans-10, cis-12 CLA had more complicated pattern.

401

However, change of amount of CLA isomers during the incubation in different heat-treated grass

402

clover concentrate confirmed that the formation of CLA isomers was greatly affected by heat

403

treatment. These results demonstrated that different heat treatments affect the formation of 19 ACS Paragon Plus Environment


Page 20 of 36

404

intermediates and/or end products in different ways, probably due to the formation of

405

intermediates and/or end products with different potencies as inhibitors of bacteria and/or

406

enzymes involved in the BH steps.56 Therefore, we speculated that changing the formation of BH

407

intermediates and/or end products may be an explanation for the different BH rates of LNA and

408

lag phase in both LA and LNA in heat-treated grass clover concentrate.

409

Although the VA formation rate was not significantly affected by the heat treatment, there was

410

an obvious numerical difference between the treatments and the highest appearance rate observed

411

for the freeze-dried grass clover concentrate. The effect of heat treatment on CLA isomers and

412

VA formation suggests an altering of bacteria which is responsible for formation of CLA isomers

413

and VA.57 In addition, the lack of effect of heat treatment on the SA formation rate demonstrates

414

that the bacteria which is responsible for the final biohydrogenation step may not be affected by

415

heat treatment.57

416

In conclusion, heat treatment has the potential of increasing rumen escape protein in green

417

protein. However, a decreased intestinal disappearance in autoclaved samples may indicate the

418

protein damage. The heat treatment would be an effective approach in reducing the LNA

419

disappearance rates in the rumen. In conclusion, the results showed that green proteins of

420

different origins response differently to the heat treatment. In addition, to prevent overheating

421

making the protein unavailable, it should be considered that green proteins are extremely heat-

422

labile.

423

Financial support

424

This Research was funded under the SPIR initiative BIOVALUE by Innovation Fund

425

Denmark. Mohammad Rashed Chowdhury had received an Erasmus-Mundus grant for his EM

426

SANF master study. 20 ACS Paragon Plus Environment

Page 21 of 36

427 428 429


Notes The authors declare no competing financial interest. Abbreviations

430

AA, amino acid; BH, biohydrogenation; CLA, conjugated linoleic acid; CP, crude protein; DM,

431

dry matter; ED, effective degradability; FA, fatty acid; LA, linoleic acid; LNA, linolenic acid;

432

LNA, linolenic acid; N, nitrogen; PUFA, poly unsaturated fatty acid; RUP, rumen undegradable

433

protein; SA, stearic acid; SCP, buffer-soluble protein; SID, small intestinal disappearance; VA,

434

vaccenic acid

435



Page 22 of 36

436

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437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480

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acids in forages. II. In vitro ruminal biohydrogenation of linolenic and linoleic acids from timothy. Canadian journal of animal science 2003, 83, 513-522. 53. Lashkari, S.; Hymøller, L.; Jensen, S. K., Ruminal biohydrogenation kinetics of defatted flaxseed and sunflower is affected by heat treatment. Journal of agricultural and food chemistry 2017, 65, 8839-8846. 54. Kim, Y. J.; Liu, R. H.; Bond, D. R.; Russell, J. B., Effect of linoleic acid concentration on conjugated linoleic acid production by butyrivibrio fibrisolvensA38. Applied and Environmental Microbiology 2000, 66, 5226-5230. 55. Loor, J.; Bandara, A.; Herbein, J., Characterization of 18: 1 and 18: 2 isomers produced during microbial biohydrogenation of unsaturated fatty acids from canola and soya bean oil in the rumen of lactating cows. Journal of Animal Physiology and Animal Nutrition 2002, 86, 422-432. 56. Gonthier, C.; Mustafa, A.; Ouellet, D.; Chouinard, P.; Berthiaume, R.; Petit, H., Feeding micronized and extruded flaxseed to dairy cows: Effects on blood parameters and milk fatty acid composition. Journal of Dairy Science 2005, 88, 748-756. 57. KEMP, P.; LANDER, D. J., Hydrogenation in vitro of α-linolenic acid to stearic acid by mixed cultures of pure strains of rumen bacteria. Microbiology 1984, 130, 527-533.



590

FIGURE CAPTIONS

591

Figure 1. Oven-dried grass clover concentrate as an example of changes in linolenic acid,

592

linoleic acid, vaccenic acid and stearic acid amounts over incubation time (g/kg DM).

593

Figure 2. Temporal changes in the disappearance and appearance of cis-9, trans-11 conjugated

594

linoleic acid of grass clover concentrate during incubations (mg/kg DM).

595

Figure 3. Temporal changes in the disappearance and appearance of trans-10, cis-12 conjugated

596

linoleic acid of grass clover concentrate during incubations (mg/kg DM).


Page 26 of 36

Page 27 of 36


Table 1. Chemical Composition of Heat Treated Green Proteina Species

Treatment

DM

Ash

CP

OM

AAb

SCP

SCP: CP

Red clover

Freeze-dried

962

281

332

719

280

58.1

0.18

Oven-dried

978

285

329

715

281

48.1

0.15

Autoclaved

933

287

338

713

270

27.2

0.08

Freeze-dried

956

159

288

841

244

25.9

0.09

Oven-dried

899

159

291

841

243

26.3

0.09

Autoclaved

900

161

295

839

238

26.3

0.09

Freeze-dried

948

82

338

918

291

58.1

0.17

Oven-dried

865

82

349

918

290

45.0

0.13

Autoclaved

880

81

348

919

281

44.4

0.13

Spin flash-dried

905

123

322

877

270

31.3

0.10

c

0.14

0.38

0.14

0.38

0.01

0.22

0.20

Ryegrass

Grass clover

P value a

Dry matter (DM), crude protein (CP, g/kg DM), organic matter (OM, g/kg DM), total amino acid (AA, g/kg DM), soluble crude

protein in a borate-phosphate buffer solution (SCP, g/kg DM), soluble crude protein in a borate-phosphate buffer solution to crude protein ratio (SCP: CP). b

Alanine+Argenine+Aspartate+Cysteine+Glutamone+Glycine+Histidine+Isoleucine+leucine+Lysine+Methinine+ornithinePheny

lalanine+Proline+Serine+Threonine +Valine. c

P value: Treatment (freeze-dried, oven-dried, autoclaved) for all species



Page 28 of 36

Table 2. Amino Acid Composition of Heat Treated Green Protein (g/kg DM)a Species

Treatment

Ala

Red

Freeze-dried

18.5

18.7

31.2

2.87

32.8

16.9

7.55

16.2

27.7

19.7

5.66

17.8

14.5

Clover

Oven-dried

18.5

19.0

31.3

2.68

33.1

17.1

7.60

16.5

27.8

19.2

5.70

17.9

Autoclaved

18.1

17.8

30.1

2.28

32.6

17.0

6.99

16.5

27.0

16.1

5.45

Freeze-dried

17.9

16.2

26.2

2.33

29.1

15.2

6.29

14.3

23.5

16.5

Oven-dried

17.9

16.2

26.3

2.21

29.1

15.3

6.16

14.4

23.6

Autoclaved

17.8

15.4

26.0

2.08

29.1

15.3

5.86

14.4

Grass

Freeze-dried

22.8

18.8

32.0

2.26

34.7

17.7

7.40

clover

Oven-dried

22.9

18.7

32.0

2.11

35.1

17.7

Autoclaved

22.3

17.9

31.0

1.90

34.2

Spin flash-dried

21.2

17.0

30.8

2.18

0.06

0.001

0.04

0.01

Ryegrass

P valueb

Arg

Asp

Cys

Glu

Gly

His

Ile

Leu

Lys

Met

Phe

Pro

Ser

Thr

Val

14.2

15.0

20.3

14.6

14.5

15.2

20.5

17.6

14.0

13.8

14.7

20.5

5.62

15.3

12.4

12.6

13.1

17.2

15.3

5.59

15.3

12.5

12.5

13.1

17.3

23.5

13.0

5.39

15.2

12.4

12.3

13.0

17.4

16.9

28.1

20.9

6.46

18.1

14.3

14.3

15.3

20.9

7.26

17.3

28.0

19.9

6.53

18.0

14.3

14.2

15.3

21.1

17.3

6.93

16.8

27.5

17.9

6.20

17.6

14.0

13.9

14.8

20.6

32.1

16.3

6.94

15.7

26.0

19.5

5.89

16.5

13.2

13.0

14.0

19.1

0.11

0.43

0.001

0.21

0.09

0.001

0.002

0.09

0.21

0.03

0.04

0.65

a

Alanine (Ala), Argenine (Arg), Aspartate (Asp), Cysteine (Cys), Glutamone (Glu), Glycine (Gly), Histidine (His), Isoleucine (Ile),

Leucine (Leu), Lysine (Lys), Methinine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Valine (Val). b

P value: Treatment (freeze-dried, oven-dried, autoclaved) for all species.


Page 29 of 36


Table 3. In Situ Dry Matter Degradation Characteristics of Heat Treated Green Proteina Species

Treatment

a

b

c

ED

Red clover

Oven-dried

70±0.01

928±0.02

4.44±0.00

504±0.03

Autoclaved

68±0.01

455±0.23

2.01±0.01

141±0.02

Oven-dried

145±0.01

859±0.01

3.10±0.00

473±0.01

Autoclaved

160±0.01

840±0.01

0.89±0.00

286±0.01

Freeze-dried

131±0.00

784±0.04

15.8±0.06

693±0.02

Oven-dried

179±0.02

818±0.02

7.25±0.00

662±0.01

Autoclaved

198±0.01

800±0.01

2.05±0.00

428±0.02

Spin flash-dried

151±0.00

749±0.01

12.5±0.02

681±0.02

P valueb

0.38

0.05

0.01

< 0.0001

P valuec

0.003

0.17

0.06

< 0.0001

Ryegrass

Grass clover

a

Soluble fraction (a, g/kg DM), potentially degradable fraction (b, g/kg DM), the rate of degradation of b fraction (c, %h-1),

effective rumen degradability calculated using a rumen fractional passage rate of 0.05 h-1 (ED, g/kg DM). Means±SEM are based on three repetitions from three individual cows of the same sample. b

P value: Treatment (oven-dried, autoclaved) × species (red clover, ryegrass, grass clover).

c

P value: Treatment (oven-dried, autoclaved, freeze-dried, spin flash-dried) for only grass clover.



Page 30 of 36

Table 4. In Situ Degradation Characteristics and Intestinal Crude Protein Disappearance of Heat Treated Green Proteina Species

Treatment

a

b

c

ED

RUP

DEP

SID

Red clover

Oven-dried

59±0.01

940±0.01

4.31±0.00

492±0.02

508±0.02

443±0.04

868±0.03

Autoclaved

57±0.01

423±0.26

1.65±0.01

105±0.02

895±0.02

68±0.03

74±0.03

Oven-dried

43±0.01

961±0.01

2.80±0.00

387±0.01

613±0.01

317±0.03

518±0.05

Autoclaved

75±0.01

925±0.01

0.66±0.00

182±0.01

818±0.01

109±0.03

133±0.04

Freeze-dried

61±0.00

920±0.06

18.9±0.08

740±0.01

260±0.01

199±0.01

762±0.02

Oven-dried

130±0.02

866±0.02

5.90±0.00

598±0.02

402±0.02

274±0.06

671±0.13

Autoclaved

114±0.00

887±0.01

1.58±0.00

326±0.02

674±0.02

145±0.03

214±0.04

Ryegrass

Grass clover

Spin flash-dried

a

115±0.00

819±0.01

9.05±0.01

637±0.02

363±0.02

238±0.03

650±0.06

b

P value

0.06

0.06

0.03

< 0.0001

< 0.0001

0.004

0.01

P valuec

0.01

0.21

0.09

< 0.0001

< 0.0001

0.07

0.003

Soluble fraction (a, g/kg DM), potentially degradable fraction (b, g/kg DM), the rate of degradation of b fraction (c, %h-1), effective rumen

degradability calculated using a rumen fractional passage rate of 0.05 h-1 (ED, g/kg CP), calculated rumen undegraded (RUP, g/kg CP), calculated digestible rumen escape protein into the small intestine of total CP intake (DEP, g/kg CP), calculated small intestinal disappearance of rumen escape protein (SID, g/kg RUP). Means±SEM are based on three repetitions from three individual cows of the same sample. b


c



Page 31 of 36


Table 5. Total Tract DM and CP Disappearance of Heat Treated Green Proteina Species

Treatment

DM

CP

Red clover

Oven-dried

853±1.98

934±1.35

Autoclaved

205 ±0.91

173±0.89

Oven-dried

604±2.30

704±3.42

Autoclaved

346±1.13

291±1.56

Freeze-dried

896±0.47

938±0.36

Oven-dried

852±3.17

871±3.89

Autoclaved

576±1.56

471±2.00

Spin flash-dried

823 ±1.27

875±1.34

P valueb

< 0.0001

< 0.0001

P valuec

< 0.0001

< 0.0001

Ryegrass

Grass clover

a

Dry matter (DM, g/kg DM), crude protein (CP, g/kg CP).

Means ± SEM are based on six repetitions from three individual cows of the same sample. b


c




Page 32 of 36

Table 6. Biohydrogenation Kinetic Parameters of Linoleic and Linolenic Acid Disappearance and Vaccenic and Stearic Acid Appearance of Heat Treated Grass Clover Measured During In Vitro Incubationsa Linoleic acid

Linolenic acid

Vaccenic acid

Stearic acid

Lagb

kc

Lag

kc

Lag

kd

Lag

kd

Freeze-dried

1.00

0.11

0.58

0.19a

0.55b

0.61

2.87

0.19

Oven-dried

2.00

0.10

1.00

0.10b

1.00b

0.12

1.33

0.11

Autoclaved

3.32

0.09

1.67

0.10b

0.37b

0.09

1.67

0.12

Spin flash-dried

3.97

0.12

2.83

0.14ab

4.33a

0.32

5.00

0.17

SEM

0.81

0.007

0.49

0.01

1.04

0.67

0.96

0.01

P value

0.68

0.48

0.44

0.04

0.009

0.33

0.59

0.47

a

Means within a column with different letters indicate significant differences (P ≤ 0.05).

b

Lag phase (hour).

c

Disappearance rate of linoleic and linolenic acid (/h).

d

Appearance rate of vaccenic and stearic acid (/h).



0.6

1.8 1.6

0.5

1.4 1.2

0.4

1.0

0.3

0.8

0.2

0.6 0.4

0.1

0.2

0.0

0.0 0

5

10

15

20

25

30

35

Incubation time (hour) Linoleic acid

Linolenic acid

Vaccenic acid

Figure 1.


Stearic acid

Stearic acid amount (g/kg DM)

Linoleic, linolenic and vaccenic acid (g/kg DM)

Page 33 of 36

cis-9, trans-11 conjugated linoleic acid (mg/kg DM)


Page 34 of 36

16 14 12 10 8 6 4 2 0 0

5

10

15

20

25

30

Incubation time (hour)

Freeze-dried

Oven-dried

Autoclaved

Spin flash-dried

Figure 2.


35


trans-10, cis-12 conjugated linoleic acid (mg/kg DM)

Page 35 of 36

7 6 5 4 3 2 1 0 0

5

10

15

20

25

30

Incubation time (hour) Freeze-dried

Oven-dried

Autoclaved

Spin flash-dried

Figure 3.


35


TOC Graphic


Page 36 of 36

Effects of Heat Treatment of Green Protein on In situ protein

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