Research Article Received: 13 December 2014
Revised: 30 April 2015
Accepted article published: 28 May 2015
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7277
Dietary supplementation of Butyrivibrio fibrisolvens alters fatty acids of milk and rumen fluid in lactating goats Swati Shivani, Anima Srivastava, Umesh K Shandilya, Vishnu Kale and Amrish K Tyagi* Abstract BACKGROUND: Conjugated linoleic acid (CLA) isomers have high health amelioration potential and hence it is of great interest to increase the CLA content in dairy products. The present study was conducted to investigate the effect of administration of high CLA producing Butyrivibrio fibrisolvens In-1 on fatty acid composition of milk and rumen fluid in lactating goats. Four groups (n = 5) of lactating goats were assigned the following treatments: Control (C) (basal diet); T1 (basal diet + linoleic acid source), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 109 CFU head−1 ) and T3 (basal diet + linoleic acid source + suspension of Butyrivibrio fibrisolvens In-1, 109 CFU head−1 ). RESULTS: Rumen liquor and milk samples were collected on days 0, 15, 30, 60 and 90 of the experiment and linoleic isomerase enzyme (LA-I) activity and fatty acid profiles were elucidated. Major effects of treatments were seen on day 30 of the experiment. Total CLA content of rumen fluid increased (P < 0.05) by 218.72, 182.26 and 304% whereas total saturated fatty acid (SFA) content was lowered (P < 0.05) by 6.1, 4.44 and 9.55% in T1, T2 and T3, respectively, as compared to control. Vaccenic acid in groups T2 and T3 increased (P < 0.05) by 66.67% and 105.7% as compared to control. In milk, total CLA increased by 2.03, 1.61 and 0.61 folds in T3, T2 and T1, respectively. Total monounsaturated fatty acid and polyunsaturated fatty acid content increased (P < 0.05) in group T3 by 14.15 and 37.44%, respectively. CONCLUSION: Results of the present study indicated that administration of B. fibrisolvens In-1 along with a linoleic acid (LA) source is a useful strategy to alter the biohydrogenation pattern in the rumen that subsequently decreased SFA content while increased CLA and unsaturated fatty acids in ruminant’s milk. © 2015 Society of Chemical Industry Keywords: fatty acids; conjugated linoleic acid; Butyrivibrio fibrisolvens; lactating goat
INTRODUCTION Over the last decade consumer’s health consciousness has become an important factor that influences the market for dairy and agro food. Healthier food products have entered the global markets with force in the past few years and rapidly gained a huge market share. However, dairy products suffer from a negative health image associated with high saturated fatty acid (SFA) content. The prospects for improving the fatty acid profile of milk and meat from ruminant animals represent a growing market for the global livestock sector as a means to support better human health. The greater proportion of SFA in ruminant products compared with other protein sources has become a subject of concern because of the potential role of dietary SFA in the aetiology of obesity, hypertension and coronary heart disease in humans.1,2 However, congujated linoleic acid (CLA), a polyunsaturated fatty acid (PUFA) present in ruminant food products is attracting considerable interest because of its diverse health beneficial outcomes in animal studies. CLA is not only a powerful anti-carcinogen, but it also has anti-atherogenic, immunomodulating, growth promoting, lean body mass enhancing and anti-diabetic properties. Experiments using several animal models indicate that dietary CLA is a J Sci Food Agric (2015)
potent nutrient partitioning agent that favours lean tissue deposition over body accretion. Therefore, ruminant nutritionists have been attempting to increase the CLA content of meat and milk by modifying feeding conditions and ruminal fermentation.3,4 It has long been known that CLA is produced by LA isomerisation in the initial step of sequential LA hydrogenation by ruminal microbes. Modifying ruminal microbial metabolism of fatty acid in rumen through animal diet formulation would be an effective way to improve the fatty acid composition of ruminant-derived foods such as milk or meat and their products to reduce saturated: unsaturated fatty acid ratio and increase CLA content. Rumen bacteria responsible for biohydrogenation of fatty acids are categories into two distinct groups (Types A and B), depending whether they are able to hydrogenate both the dienoic and
∗
Correspondence to: Amrish K Tyagi, Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal-132001, Haryana, India. E-mail: [email protected] Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal-132001, Haryana, India
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www.soci.org the monoenoic acids or only the monoenoic acid. Type A bacteria convert LA to CLA, i.e. Butyrivibrio, Micrococcus, Ruminococcus, Lactobacillus, and dominated by Butyrivibrio fibrisolvens which has higher CLA producing capacity than other ruminal bacteria. Type B bacteria produce stearate from PUFAs in rumen, i.e. Fusocillus, Clostridium proteoclasticum etc.5 Several species of CLA-producing bacteria have been isolated from the rumen, intestine and starter cultures used in the dairy industry.6 – 10 In Butyrivibrio fibrisolvens, isomerisation of LA to cis-9,trans-11-CLA is catalysed by LA isomerase (LA-I), and subsequent hydrogenation to trans-vaccenic acid (trans-VA) is catalysed by CLA reductase (CLA-R).11 Thus, if ruminal biohydrogenation of unsaturated fatty acids can be controlled, it may be possible to improve the wellness of ruminant milk and meats by increasing their unsaturated fatty acids composition in general and the n-3 fatty acids in particular. Hence, the present study has been designed to investigate the effect of administration of Butyrivibrio fibrisolvens In-1 on fatty acid composition of milk in lactating goats.
MATERIALS AND METHODS Bacterial strain and culture conditions Butyrivibrio fibrosolvens was isolated from rumen liquor of different ruminants from fistulated buffalo, cattle and goat maintained at cattle yard, NDRI, Karnal, while that of sheep were collected from slaughter house of Karnal. After collection, it was centrifuged at 2000 × g for 10 min to remove protozoa and fungi. M704 media was prepared as described by the Deutsche Sammlung von Mikroorganismen and Zellkulturen.12 The diluted rumen liquor preferably (10−5 to 10−6 dilutions) was inoculated on plates containing ATCC M704 agar media. The plates were incubated at 39 ∘ C for 48 h inside the anaerobic chambers to maintain strict anaerobic condition. The isolates were identified based on Gram staining, biochemical reactions and molecular tests. Further, Butyrivibrio fibrisolvens In-1 was found to have the highest CLA production potential in vitro among all the screened isolates. Experiment design Twenty cross-bred (Alpine × Beetal) and (Saneen × Beetal) lactating goats of 2–3 years age in their same early lactation period were selected from herd of National Dairy Research Institute, Karnal, Haryana, India. Milking was done twice daily in the morning and evening hours. On the day of sampling, the milk of both times were pooled up and transported immediately to laboratories in chilled condition. All the goats were free from physiological, anatomical, and infectious disorders. All animals were divided into four groups [control (C), T1, T2 and T3] of five animals each in a completely randomised design on the basis of their lactation yield and average body weight. The duration of the experiment was 90 days excluding 7 days as an adaptation period to the experimental diets. All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC) of National Dairy Research Institute. Feeding schedule All the goats were fed isocaloric and isonitrogenous diet as per NRC13 feeding standards. The experimental lactating goats were individually fed compound concentrate mixture formulated as per BIS type II standard and leguminous green fodder (Berseem) and the quantity of the same was adjusted at fortnightly according to body weight and milk yield. Concentrate mixture was formulated by using ingredients maize (57 parts), Ground Nut Cake (40 parts),
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mineral mixture (2 parts) and common salt (1 part). The crude protein and total lipids of the concentrate was 21.7% and 6.1%, respectively. The requirements of the goats in terms of crude protein (CP) and total digestible nutrients (TDN) under treatments were met as per NRC13 feeding standards. In addition, the goats in group II (T1) and group IV (T3) were given commercially available sunflower oil (4–10 mL day−1 ), so that the animals would receive linoleic acid at 400 mg L−1 of rumen liquor along with compound concentrate mixture up to 60 days. Moreover, the goats in group III (T2) and group IV (T3) were inoculated with an anaerobic culture of Butyrivibrio fibrisolvens In-1 (109 CFU animal−1 ), directly into the rumen through a syringe every alternate day for 30 days from the beginning of the experiment. The concentrate mixture and berseem was offered during the entire experimental period of 90 days. Collection of milk samples Milk samples were collected on days 0, 15, 30, 60 and 90. All samples were analysed for yield, total fat, solid not fat (SNF), protein and lactose content. Fatty acid analysis was done using gas chromatography as described below and total conjugated dienes were analysed through spectrophotometer. Fatty acid analysis of milk Milk samples were methylated by direct trans-esterification using the method of O’Fallon et al.14 Briefly, each milk sample (1.0 mL) was placed gently into a 16 × 125 mm screw-cap Pyrex culture tube to which 0.7 mL of 10 mol L−1 KOH in water, and 5.3 mL of methanol were added. The tube was incubated at 55 ∘ C water bath for 1.5 h with vigorous hand-shaking for 5 s every 20 min. After the tube had been cooled under tap water, 0.58 mL of 12 mol L−1 H2 SO4 was added. The tube was mixed by inversion and again incubated at 55 ∘ C in a water bath for 1.5 h with hand shaking for 5 s after every 20 min. After FAME synthesis of fatty acid methyl esters (FAME), the tube was cooled in bath of cold tap water. Hexane (3 mL) was added, and the tube was vortex-mixed for 5 min. The tube was centrifuged for 5 min in a tabletop centrifuge at 380 × g. The collected hexane was concentrated under nitrogen and stored at −20 ∘ C until GC analysis. Methyl esters were separated using gas chromatography (450-GC; Bruker, Billerica, MA, USA) equipped with a SGE Forte GC capillary column (60 m × 0.25 mm × 70 μm- BPX70). Helium was used as carrier gas at constant inlet pressure (205 kPa). The injector and detector temperature were 260 ∘ C and 270 ∘ C, respectively, and the split ratio was 1:10. The initial oven temperature was 120 ∘ C and increased by 20 ∘ C min−1 to 240 ∘ C for 55 min. The identification of individual fatty acids was based on a commercial 37 fatty acids FAME Mix (Cat # 47885; Supelco, Bellefonte, PA, USA) and CLA standard (Cat # O5507; Sigma, St Louis, MO, USA) provided with isomeric profiles in chromatogram. Assay of linoleic acid isomerase in rumen fluid LA-I assay in rumen fluid was performed by the method of Vasta et al.15 B. fibrisolvens was grown anaerobically at 39 ∘ C in ATCC-M704 medium in 12.5 × 1.5 cm culture tubes closed with screw caps fitted with butyl rubber septa (Bellco Biotechnology, Vineland, NJ, USA). Briefly, 50 mL of strained ruminal content was centrifuged at 20 000 × g for 20 min at 4 ∘ C. The supernatant was discarded, and the pellet was washed with 0.05 mol L−1 potassium phosphate buffer
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Alteration of fatty acid profile of goat milk
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(pH 6.8) and centrifuged at 20 000 g for 20 min at 4 ∘ C. Supernatant was then discarded and the microbial pellet obtained was suspended in 4 mL of 0.05 mol L−1 potassium phosphate buffer (pH 6.8). To obtain a whole-cell extract (WCE), the microbial pellet was sonicated for three cycles of 60 s each, with 90 s intervals between each cycle. An aliquot (2 mL) of the WCE was centrifuged at 20 000 × g for 15 min at 4 ∘ C and the supernatant obtained was used for microbial protein determination according to Lowry et al.16 This procedure was used to eliminate the proteins and peptides derived from feed17 and the free and bacteria membrane cell-bound tannins from the final supernatant. This procedure was previously adapted to measure microbial protein in the ruminal fluid in the presence of tannins.15 The remaining aliquot of WCE was stored at −80 ∘ C until the LA-I assay was performed as described by Vasta et al.15 with minor modifications. Briefly, 1.58 mL of 0.1 mol L−1 potassium phosphate buffer (pH 7.0) was added to 20 μL of the WCE (containing an amount of microbial protein ranging from 73.2 to 379.2 μg), followed by 300 μL of 1,3-propanediol. The reaction was initiated with the addition of 100 μL of 0.72 mmol L−1 LA in 1,3-propanediol and stopped after 4 min of incubation at room temperature by the addition of 2.5 mL of a stopper solution of isopropanol, isooctane, and 1 mol L−1 H2 SO4 (40:10:1, v/v/v). One millilitre of isooctane and 1.0 mL of water were then added, the reaction mixture was vortexed and the isooctane layer containing the fatty acids was collected. For each goat, the LA-I assay was run in duplicate. The absorbance of the isooctane layer was recorded at a wavelength of 233 nm by using a spectrophotometer, against a blank (containing 20 μL of the WCE, 1580 μL of 0.1 mol L−1 potassium phosphate buffer, pH 7.0, and 300 μL of 1,3-propanediol) in which LA was added after the stopper solution. The absorbance recorded at a 233 nm wavelength (which is the 𝜆max of the diene bonds) refers to the concentration of all the CLA isomers (CLAs’ unspecific absorbance). However, considering that in the rumen up to 90% of the conjugated linoleic acids produced from linoleic acid are represented by rumenic acid,18 we assumed that the LA-I assay can be applied primarily to the production of rumenic acid.19 Statistical analysis All data were subjected to an analysis of variance (ANOVA) according to a completely randomised design, with dietary treatments as a fixed factor, parameters observed as dependent variables. Whole data statistical calculations for five sampling times were performed as a general linear model using the univariate procedure of the software package SPSS version 17 (SPSS Inc., Chicago, IL, USA). Duncan’s method was used for multiple comparisons among means. Data from different experiments are presented graphically as mean ± SE. Differences were considered to be significant at P ≤ 0.05.
RESULTS AND DISCUSSION Milk yield and composition Milk production of experimental goats of all four groups for the 3 months was recorded and their composition was analysed. Average milk yield in different groups T1 (1.55 to 1.75 kg day−1 ), T2 (1.58 to 1.77 kg day−1 ) and T3 (1.5 to 1.68 kg day−1 ) increased until day 30 as compared to day 0, although it was not significant, and afterward it decreased gradually at days 60 and 90 (Table 1). This variation in milk yield was due to the effect of lactation progression and not because of dietary treatments. This was in agreement with results of Chiquette et al.20 J Sci Food Agric (2015)
Table 1. Effect of treatments (T1, T2 and T3) on milk yield (litres) and its composition Parameter
Days
Milk yield
Fat (%)
Protein (%)
Lactose (%)
0 15 30 60 90 0 15 30 60 90 0 15 30 60 90 0 15 30 60 90
Control 1.46 ± 0.17 1.58 ± 0.19 1.65 ± 0.18 1.49 ± 0.17 1.37 ± 0.17 3.90 ± 0.14 3.92 ± 0.27 4.05 ± 0.22 4.01 ± 0.27 3.83 ± 0.10 3.64 ± 0.07 3.40 ± 0.11 3.36 ± 0.16 3.49 ± 0.09 3.47 ± 0.10 5.13 ± 0.14 5.45 ± 0.10 5.40 ± 0.17 5.41 ± 0.07 5.25 ± 0.12
T1
T2
1.55 ± 0.12 1.66 ± 0.12 1.75 ± 0.13 1.58 ± 0.12 1.43 ± 0.12 3.73 ± 0.14 3.57 ± 0.16 3.74 ± 0.16 3.88 ± 0.13 3.82 ± 0.06 3.78 ± 0.07 3.71 ± 0.13 3.59 ± 0.06 3.57 ± 0.08 3.70 ± 0.06 5.46 ± 0.10 5.55 ± 0.09 5.52 ± 0.08 5.48 ± 0.05 5.57 ± 0.06
1.58 ± 0.17 1.67 ± 0.17 1.77 ± 0.16 1.60 ± 0.15 1.46 ± 0.15 3.72 ± 0.15 3.80 ± 0.10 3.84 ± 0.08 3.78 ± 0.08 3.90 ± 0.05 3.74 ± 0.08 3.57 ± 0.06 3.65 ± 0.07 3.59 ± 0.03 3.64 ± 0.09 5.37 ± 0.11 5.33 ± 0.15 5.37 ± 0.13 5.49 ± 0.09 5.46 ± 0.05
T3 1.50 ± 0.10 1.59 ± 0.10 1.68 ± 0.11 1.56 ± 0.08 1.42 ± 0.08 3.93 ± 0.12 3.85 ± 0.12 3.73 ± 0.11 3.88 ± 0.10 3.70 ± 0.07 3.77 ± 0.06 3.51 ± 0.09 3.71 ± 0.20 3.58 ± 0.07 3.45 ± 0.10 5.40 ± 0.08 5.28 ± 0.08 5.37 ± 0.04 5.47 ± 0.07 5.42 ± 0.08
Milk fat, protein, lactose and SNF percentage was not affected significantly by bacteria and oil administration (Table 1). These findings for fat content are comparable to the results of previous reports21 – 24 that stated no decrease in milk fat content by supplementation of polyunsaturated fatty acids (PUFAs) in diet fed to dairy goats. Schingoethe et al.25 found that milk production and energy-corrected milk were similar for cows fed yeast culture supplemented diets. The results of the present study for protein content of milk was consistent with the results obtained by Dutta and Kundu, who found that protein content in milk of cross-bred cows was not influenced by the addition of mixed probiotics culture to lactating cross-bred cows.24 Scott et al.26 also showed no added effect of yeast culture on milk lactose percentage in dairy cows. Effect on rumen microbial protein and linoleic acid isomerase activity The concentration of microbial proteins in whole cell extract (WCE) was not affected by administration of B. fibrisolvens. The administration of B. fibrisolvens In-1 resulted in higher (P < 0.05) CLA production ( μmol mL−1 min−1 ) by LA-isomerase enzyme as compared to the goats not administered with B. fibrisolvens during the assay. The LA-I specific activity ( μmol CLA min−1 mg−1 protein) was higher (P < 0.05) in both the B. fibrisolvens administered groups (683.68 and 801.59 in T2 and T3) as compared to other two non-administered groups (351.46 and 407.99) on day 30 of the experiment, in C and T1, respectively (Fig. 1). Fukuda et al.27 conducted a similar in vitro study where they found that LA-I activity of B. fibrisolvens TH1 when grown with LA was 1.8-fold higher than in the cells grown without LA. Fatty acid profile of rumen fluid The results showed that total SFA content of rumen liquor of goats in T1, T2 and T3 groups was lowered (P < 0.05) by 6.1, 4.4 and 9.6%, respectively, as compared to control on day 30 of the experiment,
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Figure 1. Effect of treatment on SFA, MUFA, PUFA and total CLA in milk of lactation goats. C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 109 CFU head−1 ) and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of B. fibrisolvens In-1, 109 CFU head−1 ). Values are means ± SE (n = 5).
whereas the SFA content of the T1, T2 and T3 groups reduced by 6.77, 2.76 and 7.29%, respectively, as compared to the control group on day 60 due to the lower content of palmitic acid (C16:0) and stearic acid (C18:0) in T2 and T3 (Table 2). However, total MUFA content increased numerically in all the groups as compared to the control on days 30 and 60 but this difference was statistically not significant. Total PUFA content increased (P < 0.05) in the T3 group at day 30 as compared to control but it was comparable to the T1 and T2 groups. However, on day 60 the total PUFA increased significantly (P < 0.05) in both bacteria-inoculated groups (T2 and T3) as compared to control. This could be due to the fact that initially inoculated bacteria were able to colonise inside the goat’s rumen for as long as 60 days but after that it started to lose its viability and the joint effect of bacteria and high LA supplement decreased. Vaccenic acid concentration increased (P < 0.05) by 66.6 and 63.11% in T2, 105.7 and 84.4% in T3 on days 30 and 60, respectively, as compared to control (Table 3). Total CLA content of rumen fluid increased by 218.72, 182.26 and 304% in T1, T2 and T3 groups, respectively, as compared to the control at day 30. Similarly at day 60, total CLA content increased to a maximum in T3 group (221.4%), followed by T1 (208.2%) and T2 (126%) as compared to control (Table 4). The level of the most biological active isomer of CLA i.e. cis-9,trans-11 CLA, in rumen fluid was 0.158, 0.366, 0.509 and 0.711 g L−1 of rumen fluid in control, T1, T2 and T3 groups, respectively, on day 30 of the experiment. The cis-9,trans-11 isomer of CLA was significantly (P ≤ 0.05) higher in T1, T2 and T3 groups as compared to control on days 30 and
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60. However, the trans-10,cis-12 isomer was significantly higher in the T3 group at day 30 and was higher in the T1, T2 and T3 groups in the day 60 rumen sample. These results highlighted that administration of B. fibrisolvens strain In-1 with dietary oil significantly reduced the total SFA content and increased total MUFA, PUFA, vaccenic acid, total CLA and isomers of CLA in rumen fluid of experimental goats. Vasta et al.19 Rana et al.28 and Miri et al.29 reported a significant reduction in SA and increase in VA with supplementation of different plant extracts which affected microbial population of group B bacteria involved in the last step of fatty acid bio-hydrogenation (VA to SA) compared to group A bacteria involved in the initial step (LA to VA).15,30 The results of present study indicated the influence of bacterial administration on rumen bio-hydrogenation and resultant higher absorption of the UFA which was further evident from the fatty acid profile of milk. However, it is clear from the data that reduction in SA content in rumen fluid was not much significant, which can be explained by the fact that in ruminants rumen microbes further reduce trans-VA to stearic acid. Fatty acid profile of milk Total SFA content decreased in the T1, T2 and T3 groups by 3.60, 4.37 and 5.6%, respectively, on day 30 of the experiment as compared to the control group, whereas on day 60 the reduction was 7.7% in the T3 group, followed by 4.79 and 3.76% in T2 and T1 as compared to control (Fig. 2A). Reduction in total SFA content was due to lower concentration of C10:0, C14:0 and C16:0, although the levels of C18:0 were unchanged. These findings were
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Alteration of fatty acid profile of goat milk
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Table 2. Effect of treatments on saturated fatty acid (SFA) content (g L−1 ) of rumen fluid Day
C
T1
0 15 30 60 90
54.081B ± 0.35 54.833AB ± 0.34 55.372aA ± 0.24 54.591aAB ± 0.34 54.818aAB ± 0.26
T2
54.371A ± 0.41 53.937A ± 0.32 51.991cB ± 0.34 50.887cB ± 0.46 53.837aA ± 0.40
54.403A ± 0.31 54.251A ± 0.35 52.913bB ± 0.05 53.078bB ± 0.34 54.074aA ± 0.35
T3 54.291A ± 0.32 53.886A ± 0.31 50.082dC ± 0.38 50.609cC ± 0.4 52.264bB ± 0.41
a,b,c Different superscript letters within rows indicate significant difference (n = 5, P < 0.05). A,B,C Different superscript letters within columns indicate significant difference (n = 5, P < 0.05).
C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil); T2 (basal diet + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ); and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ).
Table 3. Effect of treatments on vaccenic acid (g L−1 ) in rumen fluid Day
C
0 15 30 60 90
T1
T2
1.15B
1.18 ± 0.091 1.15c ± 0.047 1.23c ± 0.082 1.22b ± 0.104 1.19b ± 0.089
1.19C
± 0.090 1.30bcAB ± 0.073 1.38cA ± 0.030 1.46bA ± 0.06 1.28abAB ± 0.044
± 0.074 1.50abB ± 0.087 2.05bA ± 0.099 1.99aA ± 0.126 1.30abBC ± 0.106
T3 1.12D
± 0.101 1.64ab ± 0.104 2.53aA ± 0.083 2.25aA ± 0.089 1.50aC ± 0.120
a,b,c Different superscript letters within rows indicate significant difference (n = 5, P < 0.05). A,B,C Different superscript letters within columns indicate significant difference (n = 5, P < 0.05).
C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ) and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ).
μmol min–1 mg–1 protein
900
C
800
T1
700
T2
600
T3
500 400 300 200 100 0
0 day
15 day
30 day
60 day
90 day
Figure 2. LA-isomerase activities (μmol min−1 mg−1 protein) in rumen fluid. C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 109 CFU head−1 ) and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of B. fibrisolvens In-1, 109 CFU head−1 ). Values are means ± SE (n = 5).
The cis-9,trans-11 isomer of CLA and total CLA content was significantly increased (P < 0.05) in milk of the T3 group as compared to control, T1 and T2, whereas, in the T2 group, it was higher than control and T1 on days 30 and 60 (Fig. 2D). However, the trans-10,cis-12 isomer increased in the T3 group at day 30 only as compared to control and T1, whereas there was no statistical difference on day 60 among the groups, consistent with our results of previous studies reporting that feeding different dietary oils and probiotics significantly increased total CLA content of milk in different species of ruminants.32,35,36 The fatty acids profile in rumen fluids and milk improved with supplementation of oil (T1 group) and administration of bacteria (T2 group). The total concentration of MUFA, PUFA and CLA content was highest in the T3 group where both oil and bacteria was supplemented. Hence, these results highlight that B. fibrisolvens In-1 strain together with high LA supplement can alter the bio-hydrogenation pattern of polyunsaturated fatty acids in rumen.
CONCLUSIONS quite similar to those obtained by Nudda et al.31 and Stergiadis et al.32 where oilseed supplemented diet in goats reduced the levels of C14:0 and C16:0 in milk. In contrast, Almeida et al.33 found significant increase in the levels of C18:0 in the milk of goats fed with soybean oil supplemented diet. Total MUFA (Fig. 2B) and PUFA (Fig. 2C) content increased (P < 0.05) in the T3 group on days 30 and 60 as compared to control, T1 and T2 groups. These findings were consistent with previous reports31 – 34 where different dietary manipulations significantly increased total MUFA and PUFA content of milk from different ruminants. J Sci Food Agric (2015)
The present study concluded that administration of Butyrivibrio fibrisolvens In-1 strain alters the bio-hydrogenation of fatty acids in ruminants and manipulated ruminal microbiota to increase the vaccenic acid and PUFAs in rumen fluid, which, in turn, enhanced the beneficial fatty acid for human beings, i.e. CLA, especially cis-9,trans-11 CLA in milk. Moreover, a significant decrease in SFA and an increase in MUFA and PUFA content seemed to play a positive role in increasing this accumulation, which ultimately resulted in increased CLA content. The results in present study greatly extend the possibilities for the use of Butyrivibrio fibrisolvens In-1
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Table 4. Effect of treatments on total CLA (g L−1 ) in rumen fluid Day 0 15 30 60 90
C 0.228 ± 0.021 0.270c ± 0.029 0.203d ± 0.037 0.243c ± 0.011 0.268b ± 0.062
T1
T2
0.302C ± 0.037 0.468cBC ± 0.037 0.647cAB ± 0.044 0.749bA ± 0.046 0.479bBC ± 0.035
0.164C ± 0.008 0.397bB ± 0.011 0.573bA ± 0.028 0.55bA ± 0.039 0.421aB ± 0.028
T3 0.244C ± 0.018 0.607aB ± 0.034 0.822aA ± 0.070 0.781aA ± 0.093 0.544aB ± 0.039
a,b,c Different superscript letters within in rows indicate significant difference (n = 5, P < 0.05). A,B,C Different superscript letters within in columns indicate significant difference (n = 5, P < 0.05).
C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ) and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ).
as a potent feed additive and to increase CLA in ruminant-derived food products.
ACKNOWLEDGEMENTS The authors gratefully acknowledge the Department of Biotechnology (DBT, India) for financial support and the National Dairy Research Institute (NDRI) for providing basic infrastructure to carry out the research work.
REFERENCES 1 Baghurst K, Dietary fats, marbling and human health. Aust J Exp Agric 44:635–644 (2004). 2 Givens DI, The role of animal nutrition in improving the nutritive value of animal-derived foods in relation to chronic disease. P Nutr Soc 64:395–402 (2005). 3 Chouinard PY, Corneau L, Butler WR, Chilliard Y, Drackley JK and Bauman DE, Effect of dietary lipid source on conjugated linoleic acid concentrations in milk fat. J Dairy Sci 84:680–690 (2001). 4 Duckett SK, Andrae JG and Owens FN, Effect of high-oil corn or added corn oil on ruminal biohydrogenation of fatty acids and conjugated linoleic acid formation in beef steers fed finishing diets. J Anim Sci 80:3353–3360 (2002). 5 Griinari JM and Bauman DE, Biosynthesis of conjugated linoleic acid and its incorporation into meat and milk in ruminants, in Advances in Conjugated Linoleic Acid Research, ed. by Yurawecz MP, Mossoba MM, Kramer JKG, Pariza MW and Nelson GJ. AOCS, Illinois, pp. 180–200 (1999). 6 Kim YJ, Liu RH, Bond DR and Russell JB, Effect of linoleic acid concentration on conjugated linoleic acid production by Butyrivibrio fibrisolvens A38. Appl Environ Microbiol 66:5226–5230 (2000). 7 Coakley M, Ross RP, Nordgren M, Fitzgerald G, Devery D and Stanton C, Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species. J Appl Microbiol 94:138–145 (2003). 8 Lee SO, Kim CS, Cho SK, Choi HJ, Ji GE and Oh DK, Bioconversion of linoleic acid into conjugated linoleic acid during fermentation and by washed cells of Lactobacillus reuteri. Biotechnol Lett 25:935–938 (2003). 9 Rosberg-Cody E, Ross RP, Hussey S, Ryan CA, Murphy BP, Fitzgerald GF, et al., Mining the microbiota of the neonatal gastrointestinal tract for conjugated linoleic acid-producing Bifidobacteria. Appl Environ Microbiol 70:4635–4641 (2004). 10 Punia AK, Chaitianya S, Tyagi AK, De S and Singh K, Conjugated linoleic acid producing potential of Lactobacilli isolated from rumen of cattle. J Ind Microbiol Biochem 35:1223–1228 (2008). 11 Kepler CR, Hirons KP, McNeill JJ and Tove SB, Intermediates and products of the biohydrogenation of linoleic acid by Butyrivibrio fibrisolvens. J Biol Chem 241:1350–1354 (1966). 12 DMSZ, DMSZ 704 medium, http://www.dmsz.de/media/med 704 (1993). [15 August 2013] 13 National Research Council (NRC), Nutrient Requirement of Goats: Angora Dairy and Meat Goats in Temperate and Tropical Countries. National Academy Press, Washington, DC (1981).
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14 O’Fallon JV, Busboom JR, Nelson ML and Gaskins CT, A direct method for fatty acid methyl ester synthesis: Application to wet meat tissues, oils and feedstuffs. J Anim Sci 85:1511–1521 (2007). 15 Vasta V, Mele M, Serra A, Scerra M, Luciano G, Lanza M, et al., Metabolic fate of fatty acids involved in ruminal biohydrogenation in sheep fed concentrate or herbage with or without tannins. J Anim Sci 87:2674–2684 (2009). 16 Lowry OH, Rosebrough NJ, Farr AL and Randall RJ, Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275 (1951). 17 Makkar HPS, Dawra RK and Singh B, Determination of both tannin and protein in a tannin–protein complex. J Agric Food Chem 36:523–525 (1988). 18 Chin SF, Liu W, Storkson JM, Ha YL and Pariza MW, Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J Food Compost Anal 5:185–197 (1992). 19 Vasta V, Makkar HPS, Mele M and Priolo A, Ruminal biohydrogenation as affected by tannins in vitro. Br J Nutr 102:82–92 (2009). 20 Chiquette J, Allison MJ and Rasmussen MA, Prevotella bryantii 25A used as a probiotic in early-lactation dairy cows: effect on ruminal fermentation characteristics, milk production, and milk composition. J Dairy Sci 91:3536–3543 (2008). 21 Chilliard Y, Ferlay A, Rouel J and Lamberet G, A review of nutritional and physiological factors affecting goat milk lipid synthesis and lipolysis. J Dairy Sci 86:1751–1770 (2003). 22 Chilliard Y, Rouel J, Ferlay A, Bernard L, Gaborit P, Raynal-Ljutovac K, et al., Optimising goat’s milk and cheese fatty acid composition, in Improving the Fat Content of Foods, ed. by Williams C and Buttriss J. Woodhead Publishing, Cambridge, UK, pp. 123–145 (2006). 23 Nocek JE and Kautz WP, Direct-fed microbial supplementation on ruminal digestion, health and performance of pre- and postpartum dairy cattle. J Dairy Sci 89:260–266 (2006). 24 Dutta TK and Kundu SS, Response of mixed viable probiotics culture on milk production and nutrient availability in crossbred mid-lactating cows. Ind J Anim Sci 78:531–535 (2008). 25 Schingoethe DJ, Linke KN, Kalscheur KF, Hippen AR, Rennich DR and Yoon I, Feed efficiency of mid lactating dairy cows fed yeast culture during summer. J Dairy Sci 87:4178–4181 (2004). 26 Scott SK, Arambel MJ, Kim DY, Kent BA, Hardcastle BJ and Dawson DP, Effect of feeding yeast culture on milk production, composition, feed intake and nutrient digestibility in lactating cows. J Dairy Sci 77:300 (1994). 27 Fukuda S, Furuya H, Suzuki Y, Asanuma N and Hino T, A new strain of Butyrivibrio fibrisolvens that has high ability to isomerize linoleic acid to conjugated linoleic acid. J Gen Appl Microbiol 51:105–113 (2005). 28 Rana MS, Tyagi A, Hossain A and Tyagi AK, Effect of tanniniferous Terminalia chebula extract on rumen biohydrogenation, Δ9 -desaturase activity, CLA content and fatty acid composition in longissimus dorsi muscle of kids. Meat Sci 90:558–563 (2012). 29 Miri V, Tyagi AK, Ebrahimi HS and Mohini M, Effect of cumin (Cuminum cyminum) seed extract on milk fatty acid profile and methane emission in lactating goat. Small Rumin Res 113:66–72 (2013). 30 Kemp P and Lander DJ, Hydrogenation in vitro of alphalinolenic acid to stearic acid by mixed cultures of pure strains of rumen bacteria. J Gen Microbiol 130:527–533 (1984). 31 Nudda A, Palmquist DL, Battacone G, Fancellu S, Rassu SPG and Pulina G, Relationships between the contents of vaccenic acid, CLA and
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n-3 fatty acids of goat milk and the muscle of their suckling kids. Livestock Sci 118:195–203 (2008). 32 Stergiadis S, Leifert C, Seal CJ, Eyre MD, Steinshamn H and Butler G, Improving the fatty acid profile of winter milk from housed cows with contrasting feeding regimes by oilseed supplementation. J. Food Chem 164:293–300 (2014). 33 Almeida OC, Pires AV, Susin I, Gentil RS, Mendes CQ, Queiroz MAA, et al., Milk fatty acids profile and arterial blood milk fat precursors concentration of dairy goats fed increasing doses of soybean oil. J Small Rumin Res 114:152–160 (2013). 34 Tsiplakou E and Zervas G, The effect of fish and soybean oil inclusion in goat diet on their milk and plasma fatty acid profile. J Livestock Sci 155:236–243 (2013).
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35 Shingfield KJ, Ahvenjarvi S, Toivonen V, Vanhatalo A, Huhtanen P and Griinari JM, Effect of incremental levels of sunflower-seed oil in the diet on ruminal lipid metabolism in lactating cows. Br J Nutr 99:971–983 (2008). 36 Joy M, Ripoll-Bosch R, Sanza A, Molino F, Blasco I and Álvarez-Rodríguez J, Effects of concentrate supplementation on forage intake, metabolic profile and milk fatty acid composition of unselected ewes raising lambs. J Anim Feed Sci Technol 187:19–29 (2014).
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Revised: 30 April 2015
Accepted article published: 28 May 2015
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7277
Dietary supplementation of Butyrivibrio fibrisolvens alters fatty acids of milk and rumen fluid in lactating goats Swati Shivani, Anima Srivastava, Umesh K Shandilya, Vishnu Kale and Amrish K Tyagi* Abstract BACKGROUND: Conjugated linoleic acid (CLA) isomers have high health amelioration potential and hence it is of great interest to increase the CLA content in dairy products. The present study was conducted to investigate the effect of administration of high CLA producing Butyrivibrio fibrisolvens In-1 on fatty acid composition of milk and rumen fluid in lactating goats. Four groups (n = 5) of lactating goats were assigned the following treatments: Control (C) (basal diet); T1 (basal diet + linoleic acid source), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 109 CFU head−1 ) and T3 (basal diet + linoleic acid source + suspension of Butyrivibrio fibrisolvens In-1, 109 CFU head−1 ). RESULTS: Rumen liquor and milk samples were collected on days 0, 15, 30, 60 and 90 of the experiment and linoleic isomerase enzyme (LA-I) activity and fatty acid profiles were elucidated. Major effects of treatments were seen on day 30 of the experiment. Total CLA content of rumen fluid increased (P < 0.05) by 218.72, 182.26 and 304% whereas total saturated fatty acid (SFA) content was lowered (P < 0.05) by 6.1, 4.44 and 9.55% in T1, T2 and T3, respectively, as compared to control. Vaccenic acid in groups T2 and T3 increased (P < 0.05) by 66.67% and 105.7% as compared to control. In milk, total CLA increased by 2.03, 1.61 and 0.61 folds in T3, T2 and T1, respectively. Total monounsaturated fatty acid and polyunsaturated fatty acid content increased (P < 0.05) in group T3 by 14.15 and 37.44%, respectively. CONCLUSION: Results of the present study indicated that administration of B. fibrisolvens In-1 along with a linoleic acid (LA) source is a useful strategy to alter the biohydrogenation pattern in the rumen that subsequently decreased SFA content while increased CLA and unsaturated fatty acids in ruminant’s milk. © 2015 Society of Chemical Industry Keywords: fatty acids; conjugated linoleic acid; Butyrivibrio fibrisolvens; lactating goat
INTRODUCTION Over the last decade consumer’s health consciousness has become an important factor that influences the market for dairy and agro food. Healthier food products have entered the global markets with force in the past few years and rapidly gained a huge market share. However, dairy products suffer from a negative health image associated with high saturated fatty acid (SFA) content. The prospects for improving the fatty acid profile of milk and meat from ruminant animals represent a growing market for the global livestock sector as a means to support better human health. The greater proportion of SFA in ruminant products compared with other protein sources has become a subject of concern because of the potential role of dietary SFA in the aetiology of obesity, hypertension and coronary heart disease in humans.1,2 However, congujated linoleic acid (CLA), a polyunsaturated fatty acid (PUFA) present in ruminant food products is attracting considerable interest because of its diverse health beneficial outcomes in animal studies. CLA is not only a powerful anti-carcinogen, but it also has anti-atherogenic, immunomodulating, growth promoting, lean body mass enhancing and anti-diabetic properties. Experiments using several animal models indicate that dietary CLA is a J Sci Food Agric (2015)
potent nutrient partitioning agent that favours lean tissue deposition over body accretion. Therefore, ruminant nutritionists have been attempting to increase the CLA content of meat and milk by modifying feeding conditions and ruminal fermentation.3,4 It has long been known that CLA is produced by LA isomerisation in the initial step of sequential LA hydrogenation by ruminal microbes. Modifying ruminal microbial metabolism of fatty acid in rumen through animal diet formulation would be an effective way to improve the fatty acid composition of ruminant-derived foods such as milk or meat and their products to reduce saturated: unsaturated fatty acid ratio and increase CLA content. Rumen bacteria responsible for biohydrogenation of fatty acids are categories into two distinct groups (Types A and B), depending whether they are able to hydrogenate both the dienoic and
∗
Correspondence to: Amrish K Tyagi, Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal-132001, Haryana, India. E-mail: [email protected] Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal-132001, Haryana, India
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www.soci.org the monoenoic acids or only the monoenoic acid. Type A bacteria convert LA to CLA, i.e. Butyrivibrio, Micrococcus, Ruminococcus, Lactobacillus, and dominated by Butyrivibrio fibrisolvens which has higher CLA producing capacity than other ruminal bacteria. Type B bacteria produce stearate from PUFAs in rumen, i.e. Fusocillus, Clostridium proteoclasticum etc.5 Several species of CLA-producing bacteria have been isolated from the rumen, intestine and starter cultures used in the dairy industry.6 – 10 In Butyrivibrio fibrisolvens, isomerisation of LA to cis-9,trans-11-CLA is catalysed by LA isomerase (LA-I), and subsequent hydrogenation to trans-vaccenic acid (trans-VA) is catalysed by CLA reductase (CLA-R).11 Thus, if ruminal biohydrogenation of unsaturated fatty acids can be controlled, it may be possible to improve the wellness of ruminant milk and meats by increasing their unsaturated fatty acids composition in general and the n-3 fatty acids in particular. Hence, the present study has been designed to investigate the effect of administration of Butyrivibrio fibrisolvens In-1 on fatty acid composition of milk in lactating goats.
MATERIALS AND METHODS Bacterial strain and culture conditions Butyrivibrio fibrosolvens was isolated from rumen liquor of different ruminants from fistulated buffalo, cattle and goat maintained at cattle yard, NDRI, Karnal, while that of sheep were collected from slaughter house of Karnal. After collection, it was centrifuged at 2000 × g for 10 min to remove protozoa and fungi. M704 media was prepared as described by the Deutsche Sammlung von Mikroorganismen and Zellkulturen.12 The diluted rumen liquor preferably (10−5 to 10−6 dilutions) was inoculated on plates containing ATCC M704 agar media. The plates were incubated at 39 ∘ C for 48 h inside the anaerobic chambers to maintain strict anaerobic condition. The isolates were identified based on Gram staining, biochemical reactions and molecular tests. Further, Butyrivibrio fibrisolvens In-1 was found to have the highest CLA production potential in vitro among all the screened isolates. Experiment design Twenty cross-bred (Alpine × Beetal) and (Saneen × Beetal) lactating goats of 2–3 years age in their same early lactation period were selected from herd of National Dairy Research Institute, Karnal, Haryana, India. Milking was done twice daily in the morning and evening hours. On the day of sampling, the milk of both times were pooled up and transported immediately to laboratories in chilled condition. All the goats were free from physiological, anatomical, and infectious disorders. All animals were divided into four groups [control (C), T1, T2 and T3] of five animals each in a completely randomised design on the basis of their lactation yield and average body weight. The duration of the experiment was 90 days excluding 7 days as an adaptation period to the experimental diets. All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC) of National Dairy Research Institute. Feeding schedule All the goats were fed isocaloric and isonitrogenous diet as per NRC13 feeding standards. The experimental lactating goats were individually fed compound concentrate mixture formulated as per BIS type II standard and leguminous green fodder (Berseem) and the quantity of the same was adjusted at fortnightly according to body weight and milk yield. Concentrate mixture was formulated by using ingredients maize (57 parts), Ground Nut Cake (40 parts),
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mineral mixture (2 parts) and common salt (1 part). The crude protein and total lipids of the concentrate was 21.7% and 6.1%, respectively. The requirements of the goats in terms of crude protein (CP) and total digestible nutrients (TDN) under treatments were met as per NRC13 feeding standards. In addition, the goats in group II (T1) and group IV (T3) were given commercially available sunflower oil (4–10 mL day−1 ), so that the animals would receive linoleic acid at 400 mg L−1 of rumen liquor along with compound concentrate mixture up to 60 days. Moreover, the goats in group III (T2) and group IV (T3) were inoculated with an anaerobic culture of Butyrivibrio fibrisolvens In-1 (109 CFU animal−1 ), directly into the rumen through a syringe every alternate day for 30 days from the beginning of the experiment. The concentrate mixture and berseem was offered during the entire experimental period of 90 days. Collection of milk samples Milk samples were collected on days 0, 15, 30, 60 and 90. All samples were analysed for yield, total fat, solid not fat (SNF), protein and lactose content. Fatty acid analysis was done using gas chromatography as described below and total conjugated dienes were analysed through spectrophotometer. Fatty acid analysis of milk Milk samples were methylated by direct trans-esterification using the method of O’Fallon et al.14 Briefly, each milk sample (1.0 mL) was placed gently into a 16 × 125 mm screw-cap Pyrex culture tube to which 0.7 mL of 10 mol L−1 KOH in water, and 5.3 mL of methanol were added. The tube was incubated at 55 ∘ C water bath for 1.5 h with vigorous hand-shaking for 5 s every 20 min. After the tube had been cooled under tap water, 0.58 mL of 12 mol L−1 H2 SO4 was added. The tube was mixed by inversion and again incubated at 55 ∘ C in a water bath for 1.5 h with hand shaking for 5 s after every 20 min. After FAME synthesis of fatty acid methyl esters (FAME), the tube was cooled in bath of cold tap water. Hexane (3 mL) was added, and the tube was vortex-mixed for 5 min. The tube was centrifuged for 5 min in a tabletop centrifuge at 380 × g. The collected hexane was concentrated under nitrogen and stored at −20 ∘ C until GC analysis. Methyl esters were separated using gas chromatography (450-GC; Bruker, Billerica, MA, USA) equipped with a SGE Forte GC capillary column (60 m × 0.25 mm × 70 μm- BPX70). Helium was used as carrier gas at constant inlet pressure (205 kPa). The injector and detector temperature were 260 ∘ C and 270 ∘ C, respectively, and the split ratio was 1:10. The initial oven temperature was 120 ∘ C and increased by 20 ∘ C min−1 to 240 ∘ C for 55 min. The identification of individual fatty acids was based on a commercial 37 fatty acids FAME Mix (Cat # 47885; Supelco, Bellefonte, PA, USA) and CLA standard (Cat # O5507; Sigma, St Louis, MO, USA) provided with isomeric profiles in chromatogram. Assay of linoleic acid isomerase in rumen fluid LA-I assay in rumen fluid was performed by the method of Vasta et al.15 B. fibrisolvens was grown anaerobically at 39 ∘ C in ATCC-M704 medium in 12.5 × 1.5 cm culture tubes closed with screw caps fitted with butyl rubber septa (Bellco Biotechnology, Vineland, NJ, USA). Briefly, 50 mL of strained ruminal content was centrifuged at 20 000 × g for 20 min at 4 ∘ C. The supernatant was discarded, and the pellet was washed with 0.05 mol L−1 potassium phosphate buffer
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(pH 6.8) and centrifuged at 20 000 g for 20 min at 4 ∘ C. Supernatant was then discarded and the microbial pellet obtained was suspended in 4 mL of 0.05 mol L−1 potassium phosphate buffer (pH 6.8). To obtain a whole-cell extract (WCE), the microbial pellet was sonicated for three cycles of 60 s each, with 90 s intervals between each cycle. An aliquot (2 mL) of the WCE was centrifuged at 20 000 × g for 15 min at 4 ∘ C and the supernatant obtained was used for microbial protein determination according to Lowry et al.16 This procedure was used to eliminate the proteins and peptides derived from feed17 and the free and bacteria membrane cell-bound tannins from the final supernatant. This procedure was previously adapted to measure microbial protein in the ruminal fluid in the presence of tannins.15 The remaining aliquot of WCE was stored at −80 ∘ C until the LA-I assay was performed as described by Vasta et al.15 with minor modifications. Briefly, 1.58 mL of 0.1 mol L−1 potassium phosphate buffer (pH 7.0) was added to 20 μL of the WCE (containing an amount of microbial protein ranging from 73.2 to 379.2 μg), followed by 300 μL of 1,3-propanediol. The reaction was initiated with the addition of 100 μL of 0.72 mmol L−1 LA in 1,3-propanediol and stopped after 4 min of incubation at room temperature by the addition of 2.5 mL of a stopper solution of isopropanol, isooctane, and 1 mol L−1 H2 SO4 (40:10:1, v/v/v). One millilitre of isooctane and 1.0 mL of water were then added, the reaction mixture was vortexed and the isooctane layer containing the fatty acids was collected. For each goat, the LA-I assay was run in duplicate. The absorbance of the isooctane layer was recorded at a wavelength of 233 nm by using a spectrophotometer, against a blank (containing 20 μL of the WCE, 1580 μL of 0.1 mol L−1 potassium phosphate buffer, pH 7.0, and 300 μL of 1,3-propanediol) in which LA was added after the stopper solution. The absorbance recorded at a 233 nm wavelength (which is the 𝜆max of the diene bonds) refers to the concentration of all the CLA isomers (CLAs’ unspecific absorbance). However, considering that in the rumen up to 90% of the conjugated linoleic acids produced from linoleic acid are represented by rumenic acid,18 we assumed that the LA-I assay can be applied primarily to the production of rumenic acid.19 Statistical analysis All data were subjected to an analysis of variance (ANOVA) according to a completely randomised design, with dietary treatments as a fixed factor, parameters observed as dependent variables. Whole data statistical calculations for five sampling times were performed as a general linear model using the univariate procedure of the software package SPSS version 17 (SPSS Inc., Chicago, IL, USA). Duncan’s method was used for multiple comparisons among means. Data from different experiments are presented graphically as mean ± SE. Differences were considered to be significant at P ≤ 0.05.
RESULTS AND DISCUSSION Milk yield and composition Milk production of experimental goats of all four groups for the 3 months was recorded and their composition was analysed. Average milk yield in different groups T1 (1.55 to 1.75 kg day−1 ), T2 (1.58 to 1.77 kg day−1 ) and T3 (1.5 to 1.68 kg day−1 ) increased until day 30 as compared to day 0, although it was not significant, and afterward it decreased gradually at days 60 and 90 (Table 1). This variation in milk yield was due to the effect of lactation progression and not because of dietary treatments. This was in agreement with results of Chiquette et al.20 J Sci Food Agric (2015)
Table 1. Effect of treatments (T1, T2 and T3) on milk yield (litres) and its composition Parameter
Days
Milk yield
Fat (%)
Protein (%)
Lactose (%)
0 15 30 60 90 0 15 30 60 90 0 15 30 60 90 0 15 30 60 90
Control 1.46 ± 0.17 1.58 ± 0.19 1.65 ± 0.18 1.49 ± 0.17 1.37 ± 0.17 3.90 ± 0.14 3.92 ± 0.27 4.05 ± 0.22 4.01 ± 0.27 3.83 ± 0.10 3.64 ± 0.07 3.40 ± 0.11 3.36 ± 0.16 3.49 ± 0.09 3.47 ± 0.10 5.13 ± 0.14 5.45 ± 0.10 5.40 ± 0.17 5.41 ± 0.07 5.25 ± 0.12
T1
T2
1.55 ± 0.12 1.66 ± 0.12 1.75 ± 0.13 1.58 ± 0.12 1.43 ± 0.12 3.73 ± 0.14 3.57 ± 0.16 3.74 ± 0.16 3.88 ± 0.13 3.82 ± 0.06 3.78 ± 0.07 3.71 ± 0.13 3.59 ± 0.06 3.57 ± 0.08 3.70 ± 0.06 5.46 ± 0.10 5.55 ± 0.09 5.52 ± 0.08 5.48 ± 0.05 5.57 ± 0.06
1.58 ± 0.17 1.67 ± 0.17 1.77 ± 0.16 1.60 ± 0.15 1.46 ± 0.15 3.72 ± 0.15 3.80 ± 0.10 3.84 ± 0.08 3.78 ± 0.08 3.90 ± 0.05 3.74 ± 0.08 3.57 ± 0.06 3.65 ± 0.07 3.59 ± 0.03 3.64 ± 0.09 5.37 ± 0.11 5.33 ± 0.15 5.37 ± 0.13 5.49 ± 0.09 5.46 ± 0.05
T3 1.50 ± 0.10 1.59 ± 0.10 1.68 ± 0.11 1.56 ± 0.08 1.42 ± 0.08 3.93 ± 0.12 3.85 ± 0.12 3.73 ± 0.11 3.88 ± 0.10 3.70 ± 0.07 3.77 ± 0.06 3.51 ± 0.09 3.71 ± 0.20 3.58 ± 0.07 3.45 ± 0.10 5.40 ± 0.08 5.28 ± 0.08 5.37 ± 0.04 5.47 ± 0.07 5.42 ± 0.08
Milk fat, protein, lactose and SNF percentage was not affected significantly by bacteria and oil administration (Table 1). These findings for fat content are comparable to the results of previous reports21 – 24 that stated no decrease in milk fat content by supplementation of polyunsaturated fatty acids (PUFAs) in diet fed to dairy goats. Schingoethe et al.25 found that milk production and energy-corrected milk were similar for cows fed yeast culture supplemented diets. The results of the present study for protein content of milk was consistent with the results obtained by Dutta and Kundu, who found that protein content in milk of cross-bred cows was not influenced by the addition of mixed probiotics culture to lactating cross-bred cows.24 Scott et al.26 also showed no added effect of yeast culture on milk lactose percentage in dairy cows. Effect on rumen microbial protein and linoleic acid isomerase activity The concentration of microbial proteins in whole cell extract (WCE) was not affected by administration of B. fibrisolvens. The administration of B. fibrisolvens In-1 resulted in higher (P < 0.05) CLA production ( μmol mL−1 min−1 ) by LA-isomerase enzyme as compared to the goats not administered with B. fibrisolvens during the assay. The LA-I specific activity ( μmol CLA min−1 mg−1 protein) was higher (P < 0.05) in both the B. fibrisolvens administered groups (683.68 and 801.59 in T2 and T3) as compared to other two non-administered groups (351.46 and 407.99) on day 30 of the experiment, in C and T1, respectively (Fig. 1). Fukuda et al.27 conducted a similar in vitro study where they found that LA-I activity of B. fibrisolvens TH1 when grown with LA was 1.8-fold higher than in the cells grown without LA. Fatty acid profile of rumen fluid The results showed that total SFA content of rumen liquor of goats in T1, T2 and T3 groups was lowered (P < 0.05) by 6.1, 4.4 and 9.6%, respectively, as compared to control on day 30 of the experiment,
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Figure 1. Effect of treatment on SFA, MUFA, PUFA and total CLA in milk of lactation goats. C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 109 CFU head−1 ) and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of B. fibrisolvens In-1, 109 CFU head−1 ). Values are means ± SE (n = 5).
whereas the SFA content of the T1, T2 and T3 groups reduced by 6.77, 2.76 and 7.29%, respectively, as compared to the control group on day 60 due to the lower content of palmitic acid (C16:0) and stearic acid (C18:0) in T2 and T3 (Table 2). However, total MUFA content increased numerically in all the groups as compared to the control on days 30 and 60 but this difference was statistically not significant. Total PUFA content increased (P < 0.05) in the T3 group at day 30 as compared to control but it was comparable to the T1 and T2 groups. However, on day 60 the total PUFA increased significantly (P < 0.05) in both bacteria-inoculated groups (T2 and T3) as compared to control. This could be due to the fact that initially inoculated bacteria were able to colonise inside the goat’s rumen for as long as 60 days but after that it started to lose its viability and the joint effect of bacteria and high LA supplement decreased. Vaccenic acid concentration increased (P < 0.05) by 66.6 and 63.11% in T2, 105.7 and 84.4% in T3 on days 30 and 60, respectively, as compared to control (Table 3). Total CLA content of rumen fluid increased by 218.72, 182.26 and 304% in T1, T2 and T3 groups, respectively, as compared to the control at day 30. Similarly at day 60, total CLA content increased to a maximum in T3 group (221.4%), followed by T1 (208.2%) and T2 (126%) as compared to control (Table 4). The level of the most biological active isomer of CLA i.e. cis-9,trans-11 CLA, in rumen fluid was 0.158, 0.366, 0.509 and 0.711 g L−1 of rumen fluid in control, T1, T2 and T3 groups, respectively, on day 30 of the experiment. The cis-9,trans-11 isomer of CLA was significantly (P ≤ 0.05) higher in T1, T2 and T3 groups as compared to control on days 30 and
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60. However, the trans-10,cis-12 isomer was significantly higher in the T3 group at day 30 and was higher in the T1, T2 and T3 groups in the day 60 rumen sample. These results highlighted that administration of B. fibrisolvens strain In-1 with dietary oil significantly reduced the total SFA content and increased total MUFA, PUFA, vaccenic acid, total CLA and isomers of CLA in rumen fluid of experimental goats. Vasta et al.19 Rana et al.28 and Miri et al.29 reported a significant reduction in SA and increase in VA with supplementation of different plant extracts which affected microbial population of group B bacteria involved in the last step of fatty acid bio-hydrogenation (VA to SA) compared to group A bacteria involved in the initial step (LA to VA).15,30 The results of present study indicated the influence of bacterial administration on rumen bio-hydrogenation and resultant higher absorption of the UFA which was further evident from the fatty acid profile of milk. However, it is clear from the data that reduction in SA content in rumen fluid was not much significant, which can be explained by the fact that in ruminants rumen microbes further reduce trans-VA to stearic acid. Fatty acid profile of milk Total SFA content decreased in the T1, T2 and T3 groups by 3.60, 4.37 and 5.6%, respectively, on day 30 of the experiment as compared to the control group, whereas on day 60 the reduction was 7.7% in the T3 group, followed by 4.79 and 3.76% in T2 and T1 as compared to control (Fig. 2A). Reduction in total SFA content was due to lower concentration of C10:0, C14:0 and C16:0, although the levels of C18:0 were unchanged. These findings were
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Table 2. Effect of treatments on saturated fatty acid (SFA) content (g L−1 ) of rumen fluid Day
C
T1
0 15 30 60 90
54.081B ± 0.35 54.833AB ± 0.34 55.372aA ± 0.24 54.591aAB ± 0.34 54.818aAB ± 0.26
T2
54.371A ± 0.41 53.937A ± 0.32 51.991cB ± 0.34 50.887cB ± 0.46 53.837aA ± 0.40
54.403A ± 0.31 54.251A ± 0.35 52.913bB ± 0.05 53.078bB ± 0.34 54.074aA ± 0.35
T3 54.291A ± 0.32 53.886A ± 0.31 50.082dC ± 0.38 50.609cC ± 0.4 52.264bB ± 0.41
a,b,c Different superscript letters within rows indicate significant difference (n = 5, P < 0.05). A,B,C Different superscript letters within columns indicate significant difference (n = 5, P < 0.05).
C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil); T2 (basal diet + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ); and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ).
Table 3. Effect of treatments on vaccenic acid (g L−1 ) in rumen fluid Day
C
0 15 30 60 90
T1
T2
1.15B
1.18 ± 0.091 1.15c ± 0.047 1.23c ± 0.082 1.22b ± 0.104 1.19b ± 0.089
1.19C
± 0.090 1.30bcAB ± 0.073 1.38cA ± 0.030 1.46bA ± 0.06 1.28abAB ± 0.044
± 0.074 1.50abB ± 0.087 2.05bA ± 0.099 1.99aA ± 0.126 1.30abBC ± 0.106
T3 1.12D
± 0.101 1.64ab ± 0.104 2.53aA ± 0.083 2.25aA ± 0.089 1.50aC ± 0.120
a,b,c Different superscript letters within rows indicate significant difference (n = 5, P < 0.05). A,B,C Different superscript letters within columns indicate significant difference (n = 5, P < 0.05).
C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ) and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ).
μmol min–1 mg–1 protein
900
C
800
T1
700
T2
600
T3
500 400 300 200 100 0
0 day
15 day
30 day
60 day
90 day
Figure 2. LA-isomerase activities (μmol min−1 mg−1 protein) in rumen fluid. C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 109 CFU head−1 ) and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of B. fibrisolvens In-1, 109 CFU head−1 ). Values are means ± SE (n = 5).
The cis-9,trans-11 isomer of CLA and total CLA content was significantly increased (P < 0.05) in milk of the T3 group as compared to control, T1 and T2, whereas, in the T2 group, it was higher than control and T1 on days 30 and 60 (Fig. 2D). However, the trans-10,cis-12 isomer increased in the T3 group at day 30 only as compared to control and T1, whereas there was no statistical difference on day 60 among the groups, consistent with our results of previous studies reporting that feeding different dietary oils and probiotics significantly increased total CLA content of milk in different species of ruminants.32,35,36 The fatty acids profile in rumen fluids and milk improved with supplementation of oil (T1 group) and administration of bacteria (T2 group). The total concentration of MUFA, PUFA and CLA content was highest in the T3 group where both oil and bacteria was supplemented. Hence, these results highlight that B. fibrisolvens In-1 strain together with high LA supplement can alter the bio-hydrogenation pattern of polyunsaturated fatty acids in rumen.
CONCLUSIONS quite similar to those obtained by Nudda et al.31 and Stergiadis et al.32 where oilseed supplemented diet in goats reduced the levels of C14:0 and C16:0 in milk. In contrast, Almeida et al.33 found significant increase in the levels of C18:0 in the milk of goats fed with soybean oil supplemented diet. Total MUFA (Fig. 2B) and PUFA (Fig. 2C) content increased (P < 0.05) in the T3 group on days 30 and 60 as compared to control, T1 and T2 groups. These findings were consistent with previous reports31 – 34 where different dietary manipulations significantly increased total MUFA and PUFA content of milk from different ruminants. J Sci Food Agric (2015)
The present study concluded that administration of Butyrivibrio fibrisolvens In-1 strain alters the bio-hydrogenation of fatty acids in ruminants and manipulated ruminal microbiota to increase the vaccenic acid and PUFAs in rumen fluid, which, in turn, enhanced the beneficial fatty acid for human beings, i.e. CLA, especially cis-9,trans-11 CLA in milk. Moreover, a significant decrease in SFA and an increase in MUFA and PUFA content seemed to play a positive role in increasing this accumulation, which ultimately resulted in increased CLA content. The results in present study greatly extend the possibilities for the use of Butyrivibrio fibrisolvens In-1
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S Shivani et al.
Table 4. Effect of treatments on total CLA (g L−1 ) in rumen fluid Day 0 15 30 60 90
C 0.228 ± 0.021 0.270c ± 0.029 0.203d ± 0.037 0.243c ± 0.011 0.268b ± 0.062
T1
T2
0.302C ± 0.037 0.468cBC ± 0.037 0.647cAB ± 0.044 0.749bA ± 0.046 0.479bBC ± 0.035
0.164C ± 0.008 0.397bB ± 0.011 0.573bA ± 0.028 0.55bA ± 0.039 0.421aB ± 0.028
T3 0.244C ± 0.018 0.607aB ± 0.034 0.822aA ± 0.070 0.781aA ± 0.093 0.544aB ± 0.039
a,b,c Different superscript letters within in rows indicate significant difference (n = 5, P < 0.05). A,B,C Different superscript letters within in columns indicate significant difference (n = 5, P < 0.05).
C (Control; basal diet); T1 (basal diet + linoleic acid supplementation in terms of dietary oil), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ) and T3 (basal diet + linoleic acid supplementation in terms of dietary oil + suspension of Butyrivibrio fibrisolvens In1 @ 109 CFU head−1 ).
as a potent feed additive and to increase CLA in ruminant-derived food products.
ACKNOWLEDGEMENTS The authors gratefully acknowledge the Department of Biotechnology (DBT, India) for financial support and the National Dairy Research Institute (NDRI) for providing basic infrastructure to carry out the research work.
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