Postbiotic Production
The Lactobacillus plantarum RI11 strain was procured from the Industrial Biotechnology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia. The culture was preserved by the revival of culture following the procedure of Foo, et al.[53]. The culture was kept at −20 °C in De Man, Rogosa and Sharpe (MRS) medium (Merck, Darmstadt, Germany) with 20% (v/v) glycerol.
A volume of 100 µL from stock culture was activated in 10 mL MRS broth, incubated at 30 °C for 48 h and sub-cultured in the same media for another 24 hours. The activated culture was spread onto a plate and incubated at 30 °C for 48 h. A single colony was picked from the plate, inoculated twice into MRS broth (10 mL) and incubated at 30 °C for 48 h and 24 h. Active cells of L. plantarum RI11 was first washed using a 0.85% (w/v) NaCl (Merck, Darmstadt, Germany) sterile solution, then adjusted to 109 CFU/mL and used as an inoculum. For preparing the working culture of the L. plantarum RI11 strain, 10% (v/w) 109 CFU/mL bacterial cells were inoculated into MRS media, incubated for 10 hours at 30 °C, and centrifuged at 10,000 × g for 15 min at 4 °C. Cell-free supernatant (CFS) was filtered using a membrane of cellulose acetate (Sartorius Minisart, 0.22 µm, Gottingen, Germany) following the procedure described by Loh, et al. [54]. The harvested CFS (postbiotic RI11) was kept at 4 °C until applied in feed within 48 hours.
Ethical Note, Birds, Diets, Experimental Design and Housing
The feeding trial was performed at the research facilities of the Institute of Tropical Agriculture and Food Security (ITAFoS), Universiti Putra Malaysia. The study was conducted following the guidelines approved by Animal Ethics Committee of Universiti Putra Malaysia (protocol no. UPM/ACUC/AUP-R085/2018), which ascertains that the use and care of research animals are ethical and humane. Two hundred and fifty-two Cobb 500 male chicks (one-day-old) were supplied by a local hatchery. The chicks were housed in wire-floor cages placed in two identical rooms. The rooms were environmentally controlled with each of the two measuring 9.1 × 3.8 × 2.3 m, length × width × height, whereas measurement of each cage was 120 (length) × 120 (width) × 45 (height) cm. The birds were reared following the management recommendations of Cobb 500 from 1 to 21 days of age (starter period). The chickens in the two rooms were maintained at the recommended temperature of 32 ± 1 °C on the first day, a gradually reduced to around 24 ± 1 °C by 21 days of age. During the finisher period (day 22 to day 42), the birds were divided into 7 treatment groups, 6 replicates per group with 6 chicks in each replicate. The birds were offered 1 of 7 diets: (1) A basal diet without any supplementation as negative control (NC) 0.0% RI11; (2) NC + 0.02% (w/w) oxytetracycline as positive control (OTC); (3) NC + 0.02% (w/w) ascorbic acid as antioxidant control (AA); or four further groups were NC + 0.2, 0.4, 0.6 and 0.8 % postbiotic RI11 (v/w) of the respective levels. The basal diets were formulated using FeedLIVE software [44] following the nutrient specifications of the Cobb 500 Nutrition Guide. From day 22 to day 42, broilers were subjected to 36 ± 1 °C for 3 h per day from 11:00 am to 2:00 pm. Approximately, it took 45 min for the temperature to rise from 24 to 36 °C. Nonetheless, it took 1 h and 30 min for the temperature to decline from 36 to 24 °C. The management and environmental conditions of this current experiment were described in our companion recently published [44].
Samples Collection
At 42 days of age, around 2 hours and 30 minutes after the daily heat stress, 2 chickens from each cage (12 chickens per treatment group) were selected in random and slaughtered following the Halal procedure, as recommended in the Malaysian Standard [55]. Blood samples (exsanguination) were collected into blood tubes (BD Vacutainer®, New Jersey, USA) containing EDTA as an anticoagulant and kept on ice. Upon centrifugation at 3500 × g for 15 min at 4 °C, harvested plasma samples were (1.5 mL microcentrifuge tubes) stored at −80 °C for later determination of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) concentration. A part of the liver and ileum tissue were collected immediately after slaughtering, frozen in liquid nitrogen and stored at −80 °C for gene expression analysis including interleukin 10 (IL-10), interleukin 8 (IL-8), interleukin 6 (IL- 6), interleukin 2 (IL-2), interferon (IFN), tumor necrosis factor-alpha (TNF-α), heat shock protein 70 (HSP70), alpha 1- acid glycoprotein (α1-AGP), ceruloplasmin (CPN), zonula occludens-1 (ZO-1), mucin2 (MUC2), claudin1 (CLDN1) and occludin (OCLN) mRNA expressions.
Plasma Antioxidant Enzymes Biomarkers
Glutathione Peroxidase Activity
Glutathione peroxidase (GPx) activity was analysed in plasma samples using the EnzyChromTM Glutathione Peroxidase Assay Kit (EGPx-100, BioAssay Systems, Hayward, USA), which directly measured the consumption of NADPH in the enzyme-coupled reactions. The assay was carried out as recommended in the manufacturer's protocol. The detection range of the kit was 40- 800 U/L GPx. Approximately, 10 µL of the sample plus 90 µL of working reagent (80 µL assay buffer, 5 µL glutathione, 3 µL NADPH (35 mM), and 2 µL gr enzyme) were loaded into the microplate well and tap the plate to mix. A 100 µL of substrate solution was added to each sample and control wells. The optical density of the samples and standards were measured immediately at time zero (OD0), and again at 4 min (OD4). The absorbance of the GPx activity was recorded at 340 nm using microplate reader (Multiskan GO, Thermo Scientific, Waltham, Massachusetts, USA). The NADPH standards were used to plot the standard curve. The standard curve was used to calculate the GPx activity in the plasma samples.
Superoxide Dismutase Activity
Superoxide dismutase (SOD) assays were carried out using EnzyChrom™ Superoxide Dismutase Assay Kit (ESOD-100, BioAssay Systems, Hayward, USA) based on protocol provided from the manufacturer. The detection range of the kit was 0.05 - 3 U/mL SOD. The test depended on the addition of xanthine oxidase to the sample as a source of superoxide, and this superoxide reacted with a specific dye to form a coloured product. Based on the activity of SOD in the sample, which acted as a superoxide scavenger, the superoxide was reduced, and then the intensity of colour decreased. The activity of SOD was determined by measuring the colour intensity at 440 nm using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, Massachusetts, USA). The standard curve was used to calculate the concentration of SOD in the samples.
Catalase Activity
Catalase (CAT) activity was measured from plasma using the EnzyChromTM catalase assay kit (ECAT-100, BioAssay Systems, Hayward, USA), according to the manufacturer's protocol. The detection range of the kit was 0.2 - 5 U/L CAT. The test depended on the degradation of H2O2 using redox dye. After the preparation of the assay, 10 µL of the sample, positive control and assay buffer as blank plus 90 µL of substrate buffer (50 µM) were loaded into micro-plate well, then the plate was shaken and incubated at room temperature for 30 min. During the incubation time, the standard curve was prepared by mixing 40 µL of the 4.8 mM H2O2 reagent with 440 µL of distilled water in the serial concentration, then 10 µL of the standard solution with 90 µL of assay buffer were placed into standard wells. After incubation, 100 µL of detection reagent was combined in each well and incubated for 10 min at room temperature. Finally, the optical density of CAT was read at 570 nm using microplate reader (Multiskan GO, Thermo Scientific, Waltham, Massachusetts, USA). The standard curve was used to calculate the CAT activity in the plasma samples
Glutathione Activity
Glutathione (GSH) activity was measured in plasma using QuantiChromTM Glutathione Assay Kit (DIGT-250, BioAssay Systems, Hayward, USA) in accordance with the manufacturer's protocol. The principle of the assay depended on the reaction of 5,5'-dithiobis 2-nitrobenzoic acid with reduced glutathione to form a yellow product. Briefly, 120 µL of 20-fold diluted sample was mixed with 120 µL of reagent A into 1.5 mL tube, centrifuged at 14000 rpm for 5 min and 200 µL of supernatant was transferred into the microplate well. A 100 µL of reagent B was added to each well of samples, tapped the plate for mixing, and incubated for 25 min at room temperature. A 400 µL of the calibrator was mixed in serial dilution with distilled water into separate wells as the standard. A 300 µL of distilled water was pipetted into a separate well as a blank. After incubation, the absorbance was read at 412 nm using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, Massachusetts, USA). The GSH concentration in the plasma was calculated using the standard curve of glutathione.
RNA extraction and RT-PCR of studied genes
The extraction of total RNA from liver and ileum tissue samples were conducted using an RNeasy® Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s recommendations and protocols. A thirty mg of liver and ileum tissue samples were homogenised with 600 μL of buffer RLT and centrifuged at 4 °C, 10000 x g for 2 min to obtain the supernatant. The collected supernatant was mixed with an equal volume of 70% (v/v) undenatured ethanol. Then, RNeasy spins column was used for RNA binding and series of buffer RW1 and buffer RPE were used for RNA purification. RNase-free water was used to elute the purified RNA from the spin column. The purified RNA was confirmed for its concentration and purity using a NanodropTM 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 260/280 nm absorbance ratio. The complementary DNA (cDNA) was generated from purified RNA using a Quantitect® reverse transcription kit (Qiagen, Hilden, Germany) for quantitative PCR.
Reverse transcription real-time PCR was performance on a Bio-Rad CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA, USA). The standardisation of target genes expression was determined by the GAPDH gene as housekeeping gene. The total of 20 μL PCR reaction mixture for every sample was prepared using QuantiNova™ SYBR Green PCR kit (Qiagen, Hilden, Germany) containing 10 μL of 2X SYBR Green Master Mix, 2 μL of sample cDNA, 1 μL each of 14 μM respective forward and reverse primers and 6 μL of RNase-free water. The sequence of forward and reverse primers of target and housekeeping genes is depicted in (Table 1).
Table 1 The primer sequences of target genes used for RT-qPCR.
Target gene1
|
Primer sequence 5′-3′ 2
|
Accession No.
|
Product size (bp)
|
|
R-CTTGTGGATGGCATGATCT
|
|
|
IL-8
|
F-GCCCTCCTCCTGGTTTCA G
|
AJ009800
|
74
|
|
R-TGGCACCGCAGCTCATT
|
|
|
IL-6
|
F-GCTCGCCGGCTTCGA
|
AJ250838
|
71
|
|
R-GGTAGGTCTGAAAGGCGAACAG
|
|
IL-2
|
F-GTGGCTAACTAATCTGCTGTCCA
|
NM 204153
|
144
|
|
R-CCGTAGGGCTTACAGAAAGG
|
|
|
IL-10
|
F-TAACATCCAACTGCTCAGCTC
|
NM 001004414
|
172
|
|
R-TGATGACTGGTGCTGGTCTG
|
|
|
IFN-γ
|
F-GAGCCATCACCAAGAAGATGA
|
NM 205149
|
214
|
|
R-TAGGTCCACCGTCAGCTACA
|
|
|
TNF-α
|
F-GCTGTTCTATGACCGCCCAGTT
|
NM 204267.1
|
140
|
|
R-AACAACCAGCTATGCACCCCA
|
|
HSP70
|
F-AGCGTAACACCACCATTCC
|
NM_001006685.1
|
372
|
|
R-TGGCTCCCACCCTATCTC
|
|
|
α1-AGP
|
F-TCTGATCTAGACCTGCAGGCTC
|
AY584568.1
|
814
|
|
R-ATCCTCGCCATGGGGTTGGTG
|
|
|
CPN
|
F-GAGAGTAAGGGTGGGGGTGGG
|
XM_015291853.1
|
4134
|
|
R-TATTTCACATTTTCCACAAGG
|
|
|
MUC2
|
F-TTCATGATGCCTGCTCTTGTG
|
XM_421035
|
93
|
|
R-CCTGAGCCTTGGTACATTCTTGT
|
|
CLDN1
|
F-CATACTCCTGGGTCTGGTTGGT
|
NM_001013611.2
|
100
|
|
R-GACAGCCATCCGCATCTTCT
|
|
|
OCLN
|
F-ACGGCAGCACCTACCTCAA
|
XM_025144248
|
123
|
|
R-GGGCGAAGAAGCAGATGAG
|
|
|
ZO-1
|
F-CTTCAGGTGTTTCTCTTCCTCCTC
|
XM_015278981
|
131
|
|
R-CTGTGGTTTCATGGCTGGATC
|
|
|
GAPDH
|
F-CTGGCAAAGTCCAAGTGGTG
|
NM_204305
|
275
|
|
R-AGCACCACCCTTCAGATGAG
|
|
1 IL-8 = Interleukin 8, IL-6 = Interleukin 6, IL-2 = Interleukin 2, IL-10 = Interleukin 10, IFN-γ = Interferon gamma, TNF-α = Tumor necrosis factor alpha, HSP70 = Heat shock protein 70, α1-AGP = Alpha-1-acid glycoprotein, CPN = Ceruloplasmin, MUC2 = Mucin 2, CLDN1 = Claudin 1, OCLN = Occludin, ZO-1 = Zonula occludens-1, and GAPDH = Glyceraldehyde-3-phosphate dehydrogenase.
2 F = Forward, R = Reverse,
The real time PCR machine was programmed as follows: initial denaturation at 95 °C for 10 min, following by 40 cycles of denaturation annealing and extension (denaturation at 95 °C for 15 s, annealing at 57 °C for GADPH, HSP70, IL-10 and IFN genes, 59 °C for α1-AGP and CPN genes, 55 °C for TNF-α gene, and 60 °C for IL-6, IL-8, IL-2, OCLN, ZO-1, CLDN1, and MUC2 genes for 30 s, and finally extension at 72 °C for 20 s). Melting curve programme was included post PCR amplification cycles to confirm the amplification specificity of the primers. The efficiency of amplification of both target and housekeeping genes was performed based on standard curve of five-fold serial dilution of cDNA on a real-time PCR machine.. The relative gene expression based on housekeeping gene was quantified following the approach recommended by Livak and Schmittgen [56].
Statistical analysis
This study was subjected to a completely randomized design with the data analyses were performed using the Statistical Analysis System (SAS) 9.4 software (SAS Institute, Cary, North Carolina, USA). All data were analysed by General Linear Model (GLM) procedure of SAS and the comparison of means were identified by Duncan’s Multiple Range Test. The determination of linear and quadratic effects of increasing in inclusion of postbiotic RI11 was conducted by orthogonal polynomial contrast of SAS. The significance of statistical difference between treatments was considered at P-value < 0.05.