Subscriber access provided by CMU Libraries - http://library.cmich.edu
Article
Creating omega-3 fatty acids-enriched chicken using de-fatted green microalgal biomass Stephanie Gatrell, JongGun Kim, Theodore Derksen, Eleanore O'Neil, and Xingen Lei J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 Sep 2015 Downloaded from http://pubs.acs.org on October 16, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
Journal of Agricultural and Food Chemistry
1
Creating omega-3 fatty acids-enriched chicken using de-fatted green microalgal biomass
2
Stephanie K. Gatrell, JongGun Kim, Theodore J. Derksen, Eleanore V. O’Neil, and Xin Gen.
3
Lei*
4 5
Department of Animal Science, Cornell University, Ithaca NY 14853
6 7
⃰ Corresponding author; Tel: 607-254-4703; Fax: 607-255-9829; Email:
[email protected] 8 9
Running Title: Omega-3 enrichment with defatted microalgae
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
10
Abstract
11
This study was to create an omega-3 (n-3) fatty acids-enriched chicken product by using defatted
12
green microalgae (DGA, Nannochloropsis oceanica) biomass out of biofuel research. Hatching
13
Ross broiler chicks were fed a corn-soybean meal diet containing 0 (control), 2, 4, 8 or 16%
14
DGA for 6 wk (n = 6 cages/diet). The DGA inclusion resulted in a linear (P < 0.001) increase in
15
total n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plasma,
16
liver, breast, and thigh at wk 3 and 6. The increase in the breast EPA + DHA by the 16% DGA
17
diet reached 60-fold (P < 0.0001) over the control. The 8 and 4% DGA diets elevated (P < 0.05)
18
liver mRNA levels of ∆-9 (88%) and ∆-6 (96) desaturases. In conclusion, 8-16% of the DGA
19
can be added in diets for broilers to produce an n-3 fatty acids-enriched chicken meat.
20 21
KEY WORDS: broiler chicken, defatted microalgal biomass, docosahexaenoic acid,
22
eicosapentaenoic acid, gene expression
ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
23 24
Journal of Agricultural and Food Chemistry
Introduction Consuming diets high in long chain polyunsaturated fatty acids (PUFAs), in particular
25
omega-3 (n-3) fatty acids, has been linked to a decreased risk of cardiovascular disease, diabetes,
26
arthritis, and cancer1,2,3,4,5. However, current Western diets contain an unbalanced ratio of the
27
“pro-inflammatory” omega-6 (n-6) and “anti-inflammatory” n-3 PUFAs. These diets have an
28
average n-6:n-3 fatty acid ratio of 20-30:1, as opposed to the traditional range of 1-2:16.
29
Increasing public awareness of the health benefits of n-3 fatty acids has led researchers
30
attempting to improve the fatty acid profile of commonly consumed animal products. Because
31
the average American consumes approximately 40 kg of chicken annually7, this type of meat is a
32
promising candidate for n-3 fatty acids enrichment.
33
Fatty acid profiles of chicken breast, thigh, and skin are well-known to be highly
34
dependent on dietary fatty acid composition8. Previously, marine sources, mainly fish oil and
35
fish meal9,10,11 were used to enhance n-3 fatty acid incorporation into poultry meat. However,
36
recent increases in cost of and demand for fishmeal have led to the investigation of alternative n-
37
3 fatty acids rich sources for a more sustainable industry. Indeed, dietary inclusion of plant-
38
based α-linolenic acid (ALA)-rich flaxseed12,13,14 or canola oil15 could elevate the n-3 fatty acid
39
content of broiler meat. However, that enhancement resulted from an increased deposition of
40
ALA, not of the longer chain docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA).
41
Metabolically, ALA needs to be converted to DHA and(or) EPA to be functionally meaningful in
42
the human body. But, the efficiency of that conversion in vivo seems to be limited16.
43
Microalgae contain a superior fatty acid profile with high abundance of EPA and(or)
44
DHA to traditional animal feed protein supplements17,18,19. They also contain relatively high
45
amounts of crude protein20, essential amino acids21, and carotenoids22. Supplementation of
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
46
microalgae to poultry diets has been shown to improve the overall n-3 fatty acid status in egg
47
yolk17,23 and breast muscle16. Our laboratory has been specifically interested in defatted
48
microalgal biomass, the byproduct of biofuel research, as a partial replacement of conventional
49
poultry feed ingredients. In broiler chicks, we have demonstrated that moderate levels of
50
defatted microalgal biomass inclusion exhibited no adverse effects on growth performance or
51
health24. However, whether the defatted microalgae, with the residual fatty acids left in the
52
biomass, could enhance the deposition of n-3 fatty acids in the tissues of chicks were not
53
explored.
54
Consuming diets high in PUFAs ultimately enriches cell membranes in these fatty acids,
55
subsequently altering signaling molecules involved in carbohydrate and lipid metabolism25.
56
Enzymes involved in de novo fatty acid synthesis, such as malic enzyme (ME) and fatty acid
57
synthase (FASN) are known to be affected by dietary manipulation and feeding status26,27,28,29.
58
Furthermore, desaturase enzymes that introduce double bonds into fatty acids, including ∆-6 and
59
∆-9 desaturases, are also affected by nutritional status30,31. Thus, it is fascinating to determine
60
how the defatted microalgal biomass affects the expression of these genes and how that effect is
61
related to the enrichment of n-3 fatty acids in the chick tissues.
62
Therefore, the objectives of this experiment were: 1) to determine the feasibility in
63
producing an n-3 fatty acids-enriched chicken by feeding broiler chicks increasing levels of a
64
defatted green microalgal biomass (DGA, Nannochloropsis oceanica) (Cellana, Kailua-Kona,
65
HI), and 2) to reveal the biomass-mediated lipid metabolism gene expression mechanism related
66
to the enrichment of n-3 fatty acids in the tissues of chicks.
67 68
Materials and Methods
ACS Paragon Plus Environment
Page 4 of 28
Page 5 of 28
69
Journal of Agricultural and Food Chemistry
Animals, diets, and management: All protocols of this experiment were approved by
70
the Institutional Animal Care and Use Committee of Cornell University (IACUC approval
71
number 2010-0106). Male hatchling Ross broiler chicks were obtained from a commercial
72
hatchery and housed in a temperature-controlled unit at the Cornell University Poultry Research
73
Farm. The broiler chicks were housed in thermostatically-controlled cage batteries for the first 3
74
wk (6 chicks per cage); and 4 chicks per cage (2 killed for sample collection at wk 3) were then
75
transferred to grower cages at room temperature from wk 3 to 6. Chicks had free access to feed
76
and water and received a lighting schedule of 22 h light and 2 h dark. Birds were fed one of five
77
diets (n = 6 cages/diet) containing 0% (control), 2%, 4%, 8% or 16% DGA, on an ‘as is’ basis,
78
replacing a mixture of corn and soybean meal. Nutrient composition of DGA is shown in Table
79
S1. Starter (wk 0 to 3, Table S2) and grower (wk 3-6, Table S3) diets were formulated to be
80
isoenergetic and to meet the requirements for all essential nutrients for each phase of growth32.
81
The fatty acid profiles of each starter and grower diet are given in Table 1. At wk 3 and 6, 2
82
birds per cage were euthanized via asphyxiation with CO2 and blood was drawn from heart
83
puncture by using heparinized needles. After blood was kept on ice and centrifuged at 3000 g
84
for 15 min, plasma was stored at -20oC until analysis. Liver, breast muscle, and legs were
85
removed and a portion of each was snap frozen in liquid nitrogen and stored at -80oC until
86
analysis. Whole skinless breast and legs were sealed in plastic bags and frozen for lipid analysis.
87
Fatty acid extraction: For fatty acid extraction, experimental diets (~100 grams) were
88
ground to a fine powder. Tissue samples were taken from the liver, the core of the whole breast
89
(pectoralis major) and thigh (bicep femoris). Total lipids from the diet (100 mg), plasma (100
90
µL), and tissue samples (1 g) were extracted, with some modifications, according to Folsh et al33,
91
and were methylated with methanolic-KOH according to Ichihara et al34, by using tridecanoic
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
92
acid (Sigma-Aldrich Co., St Louis, MO) as an internal standard. Each fatty acid was identified
93
by comparing its retention time and peak area with the individual fatty acid methyl ester
94
standards (Sigma-Aldrich Co., St Louis, MO). Methyl esters of fatty acids were analyzed by
95
using a gas chromatography instrument (Agilent 6890N, Agilent Technologies, Santa Clara, CA)
96
fitted with a flame-ionization detector and used a fused-silica capillary column coated with CP-
97
SIL 88 (100 m × 0.25 mm i.d., 0.2 mm film thickness; Varian Inc, Lake Forest, CA). Oven
98
temperature was programmed to be held for 4 min at 140ºC, increased by 4ºC per min to 220ºC,
99
and then held for 5 min. Carrier gas was N2 with constant flow rate of 2 ml/sec and injector
100
temperature was 230ºC and detector temperature was 280ºC. Fatty acid content is expressed as
101
either a weight percentage of the sum of all of the fatty acids analyzed, or calculated as mg of
102
fatty acid per 100g of wet tissue.
103
Gene expression: Real time RT-PCR was performed on the snap frozen liver samples to
104
estimate the abundance of mRNA by using β-actin as a reference gene. Target genes included
105
ME, FASN, ∆-6 desaturase, and ∆-9 desaturase. After the RNA was isolated and its quality was
106
verified by agarose gel and spectrometry (A260/A280), it was transcribed by using a
107
commercially available kit (Applied Biosystems, Grand Island, NY). The resulting cDNA (0.3
108
µg) was added to a 10 µL total reaction that included Power SYBR Green PCR mater mix
109
(Applied Biosystems) and 0.625 μM forward and reverse primers (Table 2). Real-time PCR
110
analysis was performed by using a 7900HT Fast Real-Time PCR System (Applied Biosystems).
111
The PCR conditions consisted of an initial 2 minute 50oC step and a “hot start” step at 95oC for
112
10 min, followed by 40 cycles of a 95oC denaturing step for 15 sec and a 60oC annealing step
113
for 60 sec. A melting curve was analyzed for all primers to ensure the quality of the
114
amplification product. Each sample was analyzed in duplicate for both the target and reference
ACS Paragon Plus Environment
Page 6 of 28
Page 7 of 28
Journal of Agricultural and Food Chemistry
115
genes. Relative mRNA abundance was determined by using the ∆ cycle threshold (∆Ct) method.
116
For each sample, the Ct difference between the target and reference gene was calculated (∆Ct =
117
Cttarget – Ctreference). The ∆Ct values were then converted to fold differences by raising 2 to the
118
power – ∆Ct (2-∆Ct).
119
Statistical analyses: Data were pooled within cage for an experimental unit of 6 per
120
treatment, and were analyzed by ANOVA and(or) linear regression models by using PC-SAS
121
9.2. Mean differences between dietary groups were separated by using Duncan’s multiple range
122
test. For the gene expression data, selected treatment effects were directly compared with the
123
control group by using the t-test. Data are expressed as mean ± SEM, and the treatment effects
124
were deemed significant at P < 0.05, and a trend at P < 0.10.
125 126 127
Results and Discussion The animal performance and biochemical analyses, including plasma uric acid, inorganic
128
phosphorus and protein, for this experiment were reported elswhere35. Moderate levels (8%) of
129
DGA were tolerated by chicks for 6 wk without affecting growth performance or plasma
130
biochemistry. However, the higher level of inclusion (16%) led to a reduction in indices of
131
growth performance.
132
The fatty acid composition of the defatted microalgal biomass and experimental diet is
133
shown in Table 1. Containing 3.6% total lipid, the DGA was rich in EPA (representing 16.5%
134
of total fatty acids by weight), but without DHA. The fatty acid profile (as a weight percentage
135
of total fatty acids) of wk 6 plasma is shown in Table 3 (wk 3 is shown on Table S4). The main
136
fatty acid found in all dietary treatments at both time points was linoleic acid (C18:2 n6),
137
followed by palmitic acid (C16:0) and stearic acid (C18:0), which are similar. There was no
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
138
effect of DGA inclusion on saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), or
139
PUFAs, regardless of time. In contrast, the DGA inclusion resulted in a linear increase in plasma
140
n-3 fatty acids, and the increases were 5- and 15-fold by the 16% DGA diet over the control at
141
wk 3 (P < 0.0001, R2 = 0.93) and 6 (P < 0.0001, R2 = 0.75), respectively. The increases were
142
manifested with elevated EPA (C20:5 n3) and DHA (C22:6 n3), indicating that the birds were
143
able to convert the dietary EPA into DHA. At wk 6, there was a linear reduction in n-6 fatty
144
acids (P < 0.05), resulting in a favorable decrease in the ratio of n-6 to n-3 fatty acids (P
