Tailored synbiotic powder (functional food) to prevent hyperphosphataemia (kidney disorder)

Hyperphosphataemia is treated with phosphate binders, which can cause adverse effects. Spray-dried synbiotic powder (SP) composed of Lactobacillus casei JCM1134 (a phosphate-accumulating organism; PAO) and Aloe vera is potentially a safer alternative for efficient phosphate removal. In this study, a novel strategy was developed; lysine-derivatized deacetylated A. vera (DAVK) was synthesised and fabricated on phosphate-deficient PAO (PDP) for efficient phosphate transfer and then spray-dried with the supernatant of DAV centrifugation to form a sacrificial layer on PDP for SP integrity during gastric passage. In vitro experiments revealed that PAO removed only 1.6% of the phosphate from synthetic media, whereas SP removed 89%, 87%, and 67% (w/v) of the phosphate from milk, soft drink, and synthetic media, respectively, confirming the protective role of A. vera and efficient phosphate transport. Compared with commercial binders, SP effectively removed phosphate from synthetic media, whereas SP and CaCO3 exhibited comparative results for milk and soft drink. Importantly, CaCO3 caused hypercalcaemia. Thus, the described SP presents a promising tool to prevent hyperphosphataemia. This study also revealed a novel factor: diets of patients with chronic kidney disease should be monitored to determine the optimal phosphate binders, as phosphate removal performance depends on the accessible phosphate forms.


Hydrolysis methods:
Two moles TFA was used to hydrolyse Aloe vera at 121ºC for 1 h. While sulphuric acid was used at two molarities (0.5 and 1.2 M) to hydrolyse Aloe vera at 100ºC for 2 h.
Samples were neutralised before further study.

Sugar assay:
Diluted Aloe vera (300 mg) was mixed with 1 mL of 0.2% (w/v) anthrone. Sulphuric acid was heated in boiling water bath for 5 min, cooled, and the absorbance read at 620 nm 1 .
While a standard glucose estimation protocol was followed as per the Glucose assay kit (glucose CII-test, Japan).

Gas chromatography
Sample preparation: Hydrolysate samples (5 mL) were neutralised using BaCO 3 and 1 mL of 4 mg/mL methyl beta-glucoside (internal standard). Neutralised samples were filtered through filter Advantec No. 5C and the permeate was reduced with NaBH 4 at pH 8.0 and 30ºC for 1.5 h.
Amberlite resin (IR 200C) was used to convert NaBH 4 to NaBO 3 and later evaporated with methanol using a rotary vacuum evaporator. A dried sample were acetylated with 1 mL of acetic anhydride and pyridine 30ºC overnight, and later dried with a rotary vacuum evaporator. The dried sample was mixed was mixed with 0.5 mL dichloromethane and further analysed using GC. We observed different sugar content under different acid treatment and assay procedures as shown in Supplementary Table S2. Although sugar content obtained from sulphuric acid (1.2 M) and the TFA hydrolysis, using anthrone assay, were similar; sugars components differed by double. With low molarity of sulphuric acid, sugar content increased to 65 % (w/w) but higher molarity (2 M) of TFA (weaker acid) had less sugar content. We observed different sugar content under different acid treatment and assay procedures as shown in Table S2. Although sugar content obtained from sulphuric acid (1.2 M) and the TFA hydrolysis, using anthrone assay, were similar; sugars components differed by double. With low molarity of sulphuric acid, sugar content increased to 65% (w/w) but higher molarity (2 M) of TFA (weaker acid) had less sugar content (40% w/w). As per GC analysis depicted in Supplementary Table S2 and Supplementary Fig. S1, sugar in Aloe vera is composed of glucomannan where the amount of glucose is almost twice that of mannose. Still, a glucose enzymatic assay resulted in 50% (w/w) glucose in the TFA hydrolysed sample. Therefore, the exact content of sugar and its components remains unclear.
However, sugar in Aloe vera is composed of glucose and mannose in 2:1 ratio. Samples of DAV and its supernatant obtained using the deacetylation reaction of Aloe vera were freeze-dried and prepared at 0.5%, pH 6.0 in water and filtered (pore size, 0.02 µm). reported that the polysaccharides in Aloe vera ranged between 190 to 220 KDa and composed of mannose (57%), glucose (22%), and galactose (17%); while deacetylated polysaccharides in Aloe vera was of 165-185 KDa. The results of the present study differed from that of previous findings.

One factor at a time optimization for deacetylated Aloe vera-lysine (K) DAVK formation
DAV and lysine (K) were reacted at pH 8.0 and 37°C and formed lysine derivatized DAV or DAVK which was confirmed with FTIR analysis. DAVK formation is important for the competitive removal of phosphate from in vitro broth. For optimization, our target was to obtain maximum DAVK per g of DAV. Therefore, the level of DAV was kept constant (1 g). For the stability of DAV under simulated intestinal juice, pH 8.0 is important; therefore pH 8.0 was also kept constant during optimization process. The reaction temperature was kept 37°C as constant because later, PDP need to be encapsulated with DAVK at optimal temperature for the maximum PDP counts (very important for the phosphate removal performance by SP under in vitro broth) that is 37°C; additionally, at this temperature, no amino acids were degraded.

Experiment for DAVK optimization
For DAVK formation, there were two significant parameters that needed to be optimized: levels of lysine (K) and reaction time. One factor at a time approach was first applied for the different K levels: 50 mg, 100 mg, 200 mg, 400 mg, 600 mg, and 800 mg; and the other reaction parameters were 1 g DAV, pH 8.0 and volume was adjusted to 1 mL using double distilled water and vortexed, then kept at 37°C for 6 h. Samples were collected after 6 h and centrifuged at 1,915 × g for 5 min. The supernatant was analysed for free lysine using amino acid estimation 3 . As shown in Supplementary Fig. S3, among the different levels of lysine, 50 to 100 mg of lysine in reaction was found enough to completely react with 1 g of DAV as in the supernatant of reaction, the unreacted lysine concentrations were found the least. Then, we repeated the same experiment with lysine levels at 50, 60, 70, 80 and 90 mg/mL.

Initial lysine (K) in reaction (mg/mL)
Supplementary Figure S4. Determination of optimal lysine (K) level to maximize DAVK formation per g of DAV.
As shown in Supplementary Fig. S4, 50 and 60 mg/mL of lysine were completely reacted with 1 g of DAV, while 80 and 90 mg/mL of lysine were found excessive. However, 70 mg/mL of lysine was found appropriate with insignificant amount of unreacted lysine that shows the enough amount of lysine was used to react with 1 g of DAV completely. Therefore, 70 mg/mL of lysine was found optimal for the optimization of DAVK formation from 1 g of DAV.
Reaction time was optimized for the maximization of DAVK formation by analysing the unreacted lysine in reaction mixture. The unreacted lysine levels were evaluated at different reaction time (1, 2, 3, 4, 5 and 6 h) under the following reaction conditions: 1 g DAV and 70 mg lysine were mixed, and volume was adjusted to 1 mL using double distilled water, then As shown in Fig. 2, 50 and 60 mg/mL of lysine were completely reacted with 1 g of DAV, while 80 and 90 mg/mL of lysine were found excessive. However, 70 mg/mL of lysine was found appropriate with insignificant amount of unreacted lysine that shows the enough amount of lysine was used to react with 1 g of DAV completely. Therefore, 70 mg/mL of lysine was found optimal for the optimization of DAVK formation from 1 g of DAV.
Reaction time was optimized for the maximization of DAVK formation by analysing the unreacted lysine in reaction mixture. The unreacted lysine levels were evaluated at different reaction time (1, 2, 3, 4, 5 and 6 h) under the following reaction conditions: 1 g DAV and 70 mg lysine were mixed, and volume was adjusted to 1 mL using double As shown in Supplementary Fig. S5, the significance levels of reaction time data at different time intervals merged at and beyond 3 h. This result concluded the three hour of reaction time was suitable since further time elongation did not result in significant data or lower unreacted lysine in reaction mixture.
The optimized parameters of DAVK formation were as follows: 1 g DAV and 70 mg lysine were mixed, and volume was adjusted to 1 mL using double distilled water, then vortexed at pH 8.0, and kept at 37°C for 3 h of reaction time.