A. bilimbi fruit (ripe condition) was picked from the campus gardens, while shrimp paste was acquired from Rembang Regency, Central Java Province, Indonesia. The conventional plate counting method using de Man Rogosa and Sharpe’s agar (MRS; Merck KGaA, Darmstadt, Germany) cultured anaerobically at 38°C for 48 h, shrimp paste contained 14.15 ± 0.33 log CFU/gm. Mareta et al. [13 (link)] described the fermentation of A. bilimbi fruit filtrate with shrimp paste. Briefly, A. bilimbi fruit was washed and drained before being crushed with an electric blender and filtered through a cheese cloth. The shrimp paste was then inoculated (gm:l) into the prepared fruit filtrate (pH 1.45 ± 0.06, determined with a portable pH meter, OHAUS ST300) in an anaerobic jar and left at room temperature. The FF had LAB counts of 30.37 ± 0.16 log CFU/ml and a pH of 1.30 ± 0.08 after 4 days of incubation. The fermented products were kept at −10°C until utilized in in vivo studies.
St300
Manufactured by Ohaus
Sourced in United States
The ST300 is an analytical balance from Ohaus designed for laboratory use. It has a maximum capacity of 300 grams and a readability of 0.001 grams. The balance features a stainless steel weighing platform and a backlit LCD display.
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5 protocols using st300
1
Fermentation of Averrhoa bilimbi fruit with shrimp paste
2
Biochemical Analysis of Microbial Cultivation
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Changes in pH during the cultivation were monitored using a pH meter (ST300, OHAUS, Parsippany, NJ, USA). Prior to biochemical analysis, samples were centrifuged at 1000× g for 10 min at room temperature. Then, the supernatant was collected into a new tube, and the process was repeated until no pellet was visually observed. Sugars (glucose and fructose) were measured using the K-SUFRG kit, ethanol was measured using K-ETOH kit, and acids (gluconic and glucuronic acids) were measured using K-GATE and K-URONIC kits (Megazyme, International Ireland Ltd., Bray, Ireland), according to the manufacturer’s instructions.
Free amino nitrogen (FAN) concentration was measured using the ninhydrin analysis method [21 ]. The ninhydrin color reagent consisted of 0.3 g fructose, 6 g KH2PO4, 10 g Na2HPO4 and 0.5 g ninhydrin dissolved in 100 mL of distilled water. A solution mixture was prepared by dissolving 2 g of potassium iodide into 600 mL of distilled water and 400 mL of 96% ethanol. The samples were first diluted 50 times using distilled water. Then, 2 mL of the diluted sample was mixed with 1 mL of the ninhydrin color reagent. The mixture was boiled for 16 minutes and left to cool down to room temperature using an ice bath for 20 min. Afterwards, 5 mL of the previously prepared solution mixture was added to each sample, and the absorbance was measured at 570 nm.
Free amino nitrogen (FAN) concentration was measured using the ninhydrin analysis method [21 ]. The ninhydrin color reagent consisted of 0.3 g fructose, 6 g KH2PO4, 10 g Na2HPO4 and 0.5 g ninhydrin dissolved in 100 mL of distilled water. A solution mixture was prepared by dissolving 2 g of potassium iodide into 600 mL of distilled water and 400 mL of 96% ethanol. The samples were first diluted 50 times using distilled water. Then, 2 mL of the diluted sample was mixed with 1 mL of the ninhydrin color reagent. The mixture was boiled for 16 minutes and left to cool down to room temperature using an ice bath for 20 min. Afterwards, 5 mL of the previously prepared solution mixture was added to each sample, and the absorbance was measured at 570 nm.
3
Comprehensive Characterization of VP-TGMS Hydrogels
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Scanning electron microscopy (SEM) measurements were performed using an FEI nanoSEM NOVA230 microscope. VP-TGMS gels were generated by lyophilization, and the morphology of the samples was characterized by SEM at an operating voltage of 5 kV. The rheological properties of the injectable VP-TGMS hydrogels were explored using a Thermo Fisher Scientific rheometer (HAAKE MARS III) with a 20-mm parallel plate. The frequency for the strain sweep and time sweep measurements was set to 10.0 rad/s. The pH values were detected in the micellar solution before VP-TGMS became a gel state using a handheld pH meter (OHAUS ST-300). The osmolarity of the VP-TGMS gels was tested with a colloid osmometer (OSMOMAT 050). The recording system was calibrated prior to each experiment. Swelling studies were carried out to assess the hydration capacity of the VP-TGMS gels at 37 °C. The initial mass of the VP-TGMS hydrogel (W0) was recorded. Subsequently, the VP-TGMS hydrogels were immersed in PBS. The mass of swollen hydrogels in equilibrium at each time point (Wt) was then measured. Three samples were used at each time point in the study. The swelling ratio was calculated using the following equation: swelling ratio = (Wt − W0)/W0.
4
Measuring pH and Total Acidity
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The pH values of the samples were measured with a pH meter (ST300, Ohaus, USA). Total acidity was determined by volumetric analysis method using 0.1 N NaOH solution, 10 mL of sample, and phenolphthalein solution as the indicator, and the results were calculated as g acetic acid/100 mL (Vitas, Cvetanovıć, Maškovıć, Švarc-Gajıć, & Malbaša, 2018 (link)).
5
In Vitro Protein Digestibility Determination
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The method of López-Monterrubio et al. (2020) (link) was used for the determination of IVPD of YP and four plant protein samples, using a multi-enzyme system included 1.6 mg trypsin (14,600 U/mg), 3.1 mg α-chymotrypsin (48 U/mg) and 1.3 mg peptidase (102 U/g) per mL. 50 mL of protein suspensions (1 mg N/mL) were prepared and use a magnetic stirrer to disperse them evenly. Subsequently, the suspensions were incubated at 37 °C for 10 min and the pH was adjusted to 8.0 by using 0.2 M NaOH. Five milliliters of the multi-enzyme solution were then added to each protein suspension which was kept at 37 °C in a water bath shaker and the pH was recorded by a pH meter (ST300, OHAUS, USA) after 10 minutes. The percent IVPD was calculated by the following equation: Where: pH10min is the pH value of the solution measured by pH meter after 10 min of enzymatic digestion.
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