Materials
Streptozotocin (STZ; catalogue number ALX-380-010-G001) was purchased from Enzo Life Science (NY, USA). Psicose was from Merck (Darmstadt, Germany; catalogue number P8043). L.plantarum IS-10506 was cultured and prepared as described before (16).
In vivo animal trial
Animals and housing
Because in Indonesia only the Ministry of Health and universities with a medical faculty have ethical committees (and this excludes the universities participating in this study), the ethical committee of the Faculty of Medicine of the University of Indonesia (the most reputable committee) was chosen to assess the animal trial. All animal care procedures were conducted under the animal protocol approved by the ethical committee (Ref:1196/UN2.F1/ETIK/2018) under approval number 18-09-1045. Male Sprague Dawley rats were purchased from the Animal Experimental Laboratory National Agency of Drug and Food Control (Jakarta, Indonesia) at 6 weeks of age and were allowed to adapt for 14 days. They were housed in individual cages and maintained with a 12-hour light/dark cycle, at 21–23 °C and 55%±5% humidity. All rodents were given ad libitum access to water and commercially available rat normal pellet diet (NPD) purchased from local market, during a 14 days acclimatization period, prior to the dietary manipulation. Subsequently, purified Rodent Diet AIN-93M, a modified AIN-76A standard diet (American Institute of Nutrition) (26) was provided to the rats as a control.
Diets
Native taro starch ”HASIL BUMIKU” was was purchased from a local supplier (Kusuka Ubiku) in Bantul, Yogyakarta, Central Java, Indonesia. Modified taro-starch was manufactured by the autoclave-cooling method according to a modification of the method of Zhao and Lin (27). In brief, taro starch was blended with distilled water based on the ratio 1:3.5, and the blend was then gelatinized using pressure-heated instrument at 121 °C for 30 minutes and cooled 4 °C, with a repetition of two cycles. Afterwards, the retrogradaded starch was dried using a fan-assisted oven at 60 °C for 16 hours after which it was allowed to cool at room temperature for 24 hours, and subsequently grinded, and sieved using a 60 mesh.
Beet juice was adsorbed to both native and modified taro-starch by absorbing beetroot juice, at a ratio of 1:1, and then drying in an oven at 40 °C for 16 hours. Beet juice was adsorbed to both native and modified taro starch by absorbing beetroot juice, at a ratio of 1 : 1, and then drying in an oven at 40 °C for 16 hours. These were prepared and fed according to the dose of beet juice of 6 ml/day, but in adsorbed beet juice form.
Beet juice (6 ml/day) was also provided separately, as well as in combination with native and modified starch (non-adsorbed). The probiotic L.plantarum IS-10506 was given by gavage at 1010 colony forming units/day.
Rats were first provided modified AIN-93M by replacing corn starch with the respective taro starch, and replacing sucrose and cellulose with maltodextrin. This ration was provided for 3 days, with 25% incremental increasing dose of the modified AIN (at 75, 50 and 25% commercial rat pellet diet and 25, 50, and 75% dietary intervention formulation for the 3 days, respectively). Beet juice and probiotic were added to AIN-93M diet where applicable by gavage.
Development of type 2 diabetes by STZ-treatment
Four rats in each group were allocated to the dietary treatments. Then the rats were injected intraperitoneally (i.p.) with 120 mg kg-1 nicotinamide in 0.9 % NaCl, followed after 15 minutes by STZ in citrate buffer pH 4.4 (70 mg kg−1), and four days after the STZ injection, fasting and postprandial blood glucose levels were measured using a Freestyle glucose meter (Easy Touch GCU 3 in 1) from a puncture at the tip of the tail. The rats with a fasting glucose of ≥100 mg dl-1 and/or postprandial blood glucose levels of ≥140 mg dl−1 were considered as type 2 diabetic, and those rats which had not yet developed T2D within these 4 days were injected for the second time with 120 mg kg-1 nicotinamide and STZ in citrate buffer pH 4.4 (70 mg kg−1).
After confirmation of T2D, the eight week old rats of 190 - 220 g were divided into 10 groups of n=4 each, namely:
- AIN-93M (control),
- AIN-93M with two times in a day 3 ml psicose by gavage (psicose),
- AIN-93M with two times in a day 3 ml beetroot juice by gavage (beet juice),
- native taro starch (native starch),
- modified taro starch (modified starch),
- native taro starch with beetroot juice adsorbed (native beet adsorbed),
- modified taro starch with beetroot juice adsorbed (modified beet adsorbed),
- native taro starch combined with two times in a day 3 ml beetroot juice by gavage (native + beet),
- modified taro starch combined with two times in a day 3 ml beetroot juice by gavage (modified + beet), and
- plantarum IS-10506 (probiotic).
The rats were allowed to continue to feed on their respective diets until the end of the study. Food and water intake were monitored every day. Bodyweight was monitored weekly. The average food intake per rat was calculated. Fecal pellets were collected at the end of one week of feeding, as well as after 4 weeks.
Extraction of nucleic acids
DNA of feces samples was performed using the Quick-DNA™ Fecal/Soil Microbe Miniprep Kit (Zymo Research) according to manufacturer’s instructions, using the Precellys 24 tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France), applying 3 cycles of 30 seconds each, with 5 minute cooling on ice in between.
PCR-amplifying the V3-V4 region of the 16S rRNA gene and next generation sequencing
Illumina 16S rRNA gene amplicon libraries were generated and sequenced at BaseClear (Leiden, the Netherlands). In short, barcoded amplicons from the V3-V4 region of 16S rRNA genes were generated using a 2-step PCR. 10-25 ng isolated genomic DNA was used as template for the first PCR with a total volume of 50 μl using the 341F (5’-CCTACGGGNGGCWGCAG-3’) and the 785R (5’-GACTACHVGGGTATCTAATCC-3’) primers appended with Illumina adaptor sequences (Illumina, San Diego, CA, USA). PCR products were purified and the size of the PCR products were checked on Fragment analyzer (Advanced Analytical Technologies, Heidelberg, Germany) and quantified by fluorometric analysis. Purified PCR products were used for the 2nd PCR in combination with sample-specific barcoded primers (Nextera XT index kit, Illumina). Subsequently, PCR products were purified, checked on a Fragment analyzer (Advanced Analytical Technologies) and quantified, followed by multiplexing, clustering, and sequencing on an Illumina MiSeq with the paired-end (2x) 300 bp protocol and indexing.
Sequence processing and analyses
The sequencing run was analyzed with the Illumina CASAVA pipeline (v1.8.3) with demultiplexing based on sample-specific barcodes. The raw sequencing data produced was processed removing the sequence reads of too low quality (only "passing filter" reads were selected) and discarding reads containing adaptor sequences or PhiX control with an in-house filtering protocol. A quality assessment on the remaining reads was performed using the FASTQC quality control tool version 0.10.0. Subsequently, the sequences were further analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) software pipeline, version 1.9.1 (28) for α- and β-diversity and (un)weighted principal coordinate analysis. The software package R (3.5.0) (R Core Team, 2013) was used to determine correlations between OTUs and treatments. Statistical analyses were performed with RStudio (1.0.153). Kruskal-Wallis correlations were calculated between OTUs and non-continuous values (treatments). Multiple comparison was corrected using the false discovery rate (FDR), and q-values (adjusted p-values) were considered significantly different at q <0.05.
Data availability
The datasets are available from the corresponding author on reasonable request. Raw sequences have been deposited in the European Nucleotide Archive under submission number PRJEB39722: (https://www.ebi.ac.uk/ena/browser/view/PRJEB39722).