Indian gooseberry, or amla (Phyllanthus emblica L.), is a nutrient-dense fruit prized for its strong antioxidant qualities and high concentration of vitamin C, polyphenols and flavonoids (Raju et al., 2025). Amla is widely grown in India, with notable production in places like Madhya Pradesh, Tamil Nadu and Uttar Pradesh, which makes it available for a variety of industrial uses (Tewari et al., 2024).
       
Fruit juices, including amla juice, have traditionally been processed using thermal techniques like pasteurization to guarantee their microbiological safety and shelf stability. However, these techniques frequently cause heat-sensitive compounds like polyphenols and ascorbic acid to degrade, thus impacting the juice taste and nutritional value (Bhattacherjee et al., 2011). The capacity of non-thermal processing methods to inactivate bacteria and enzymes while maintaining the sensory as well as nutritional qualities of food products has drawn attention as a solution to these problems. Ultrasound-assisted processing has become one of the most promising among these. Ultrasound uses high frequency sound wave to cause cavitation, which disrupt cells and enhanced mass transfer, which can improve bioactive compounds extraction and inactivate spoilage enzymes without significant heat generation (Tewari et al., 2023).
       
Effectiveness of ultrasound processing in improving amla juice quality has been shown in recent studies. For example, it has been demonstrated that ultrasound treatment greatly inactivates the enzymes peroxidase (POD) and polyphenol oxidase (PPO), which cause enzymatic browning and improve the juice’s colour stability. Furthermore, compared to traditional thermal treatments, ultrasound processing has been shown to maintain larger amounts of ascorbic acid and boost the extraction yield of phenolic compounds.
       
Previous studies on ultrasound-assisted processing have largely focused on other fruit matrices and general quality parameters, leaving a clear research gap regarding amla (Phyllanthus emblica L.) juice, as the effects of different ultrasound amplitudes on biochemical stability, colour attributes and especially microbial inactivation have not yet been systematically investigated.
       
Given these advantages, optimizing ultrasound-assisted processing parameters is crucial for retention of bioactive compounds and ensure the microbial safety of amla juice. This study aims to discover the effects of ultrasound processing on the biochemical, microbial and colour attributes of amla juice and to determine the optimal processing conditions that preserve its nutritional and sensory qualities (Tsai et al., 2014).

Materials and chemicals
 
Fully matured NA-7 variety of Indian gooseberry (Amla) was sourced from the Horticulture Farm of the College of Horticulture, Anand Agricultural University, Gujarat, India. Reagents and chemicals such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), sodium hydroxide, methanol, L-ascorbic acid, ethanol, metaphosphoric acid, sodium nitrate, sodium salt of 2,6-dichlorophenol indophenol, sodium bicarbonate, sodium carbonate, gallic acid, Folin-Ciocalteu reagent, quercetin, aluminium chloride, hydrochloric acid, tartaric acid, violet red bile agar (VRBA), nutrient agar (NA) and potato dextrose agar (PDA) were acquired from HiMedia Laboratories Pvt. Ltd., Mumbai, Maharashtra, India.
 
Preparation of amla juice
 
Fresh Amla fruits were thoroughly washed, deseeded and cut into small segments. These pieces were then pulped using a commercial juicer (JAS Enterprises, Ahmedabad, Gujarat). The resulting juice was filtered using a muslin cloth and stored under refrigeration until further processing. Juice samples were packed in 100 mL polyethylene terephthalate (PET) bottles.
 
Ultrasound processing of amla juice
 
Ultrasound treatment of amla (Phyllanthus emblica. L) juice was carried out using a Vibra-Cell Ultrasonic processor (Sonics and Materials Inc., USA) at a frequency of 20 kHz with a maximum power output of 130 W. The juice samples were sonicated at varying amplitudes (75% to 95%) for 5 to 15 minutes, depending on the experimental design. The sonication was applied in pulsed mode, with 15 seconds of active operation followed by 5 seconds of rest, to minimize excessive heat generation and preserve heat-sensitive nutrients. The temperature is maintained at 20±2°C using ice bath.
 
Microbiological evaluation
 
Coliform level,yeast and mold count and total plate count were calculated using pour plate method to asses the microbial quality. Nutrient agar was used for total plate count (incubated at 37°C for 24-48 h), VRBA was used for coliform analysis (incubated at 37°C for 24-48 h) and PDA was used for yeast and mold (count incubated at 25°C for 48-72 h), (Ranganna, 1986).
 
Estimation of ascorbic acid
 
Estimation carried out using titration method. 10 mL juice sample was diluted to 100 mL using 3% metaphosphoric acid, after filtering the mixture using Whatman No.1 filter paper, 10 mL of extract was titrated against 2,6-dichlorophenol indophenol until a pink endpoint emerged (Ranganna, 1986). The ascorbic acid content was calculated using the formula:
 

 

 
Where,
T= Titration volume.
D= Dye factor.
V= Total volume.
A= Aliquot volume.
W= Sample weight.
 
Antioxidant activity
 
Radical scavenging activity of the juice was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. To prepare sample, 1.0 g of juice was homogenized in 10 mL of methanol, followed by centrifugation at 4000 rpm for a duration of 10 minutes.
       
For the reaction, a 100 µL aliquot of the supernatant was introduced to 2.9 mL of methanolic solution containing 0.1 mM DPPH. This mixture was kept in the dark for 30 minutes for incubation. The resulting absorbance was quantified at 517 nm using a spectrophotometer, following the protocol established by Ranganna (1986). Antioxidant activity (%) was calculated as:
 

 

 
Where,
AB= Control absorbance.
AA= Sample absorbance.
 
pH determination
 
Digital pH (Chemi line digital pH meter, CL 120) was used to monitor pH.
 
Total soluble solids (TSS)
 
Digital hand-held refractometer was used to quantify TSS at 25±3°C. Double-distilled water was used for calibration.
 
Titratable acidity (TA)
 
TA was expressed as % of citric acid. After diluting a 10 mL juice sample to 250 mL with distilled water, 100 mL of this was titrated using phenolphthalein indicator against 0.1 N NaOH (Ranganna, 1986). The percentage of citric acid was calculated as:
 

  

 
Where,
T= Titration volume.
N= Normality of NaOH.
V= Total volume.
E= Equivalent weight of citric acid.
W= Sample weight.
V1= Aliquot volume.
 
Colour analysis
 
A lovibond tintometer was used to measure the color parameters L (lightness), a (redness) and b (yellowness) at 25±3°C. Each measurement was replicated three times. Total colour difference (DE) was computed as:
 

 

Where,
L, a, b= Sample values.
L^o, a^o, b^o = Control values of the untreated juice.
 
Total phenolic content
 
Folin-Ciocalteu method was used to estimate phenolic content. One mL of sample was extracted in 20 mL of 80% methanol for 4 hours at 37°C, further the content was centrifuged at 4000G for 10 min. A 60 µL aliquot was reacted sequentially with 1 mL water, 100 µL FC reagent, 490 µL water and 400 µL 20% sodium carbonate. Subsequently 30 minutes of incubation at 27°C, absorbance was measured at 750 nm. The results were given in milligrams of gallic acid equivalents per 100 grams of fresh weight (Ranganna, 1986).
 
Total flavonoid content
 
A colorimetric test was used to quantify flavonoids. The process of extraction was comparable to that of phenolics. A 1 mL aliquot was filled with 0.3 mL of 5% sodium nitrate and 4 mL of water. After five minutes, 0.3 mL of 10% aluminum chloride and after six minutes two milliliters of 1M NaOH were added. The final absorbance was measured at 510 nm following a 30-minute incubation period at 27°C. The results were expressed as milligrams of quercetin equivalents for every 100 grams of fresh weight (Ranganna, 1986).
 
Statistical analysis
 
Analytical tests were carried out three times. The mean ± standard deviation was used to express the results. GraphPad Prism 5 was used for statistical analysis (significance level α = 0.05). Microsoft Excel 2010 was used for ANOVA, correlation analysis and linear modeling. Response surface methodology (RSM) in Design-Expert 13 software was used to optimize ultrasound processing parameters. The optimization findings were validated by comparing the experimental results with the values predicted by the model.

Optimization of ultrasound processing
 
Current study evaluated the optimization of ultrasound processing parameters amplitude and time for enhancing the microbial safety, nutritional value and sensory quality of amla (Phyllanthus emblica. L) juice. Statistical modeling and ANOVA revealed that all developed models for microbial quality, ascorbic acid, antioxidant activity and colour value were significant, with high R² values (0.960-0.989), indicating good predictive accuracy. Microbial reduction was significantly influenced by amplitude (F = 490.38), time (F = 93.40) and their interaction, with higher amplitude and time reducing microbial load due to cavitation effects, which disrupt microbial cell walls (Tiwari et al., 2008). Ascorbic acid content was strongly affected by amplitude and its quadratic term (F = 157.62), with moderate ultrasound preserving higher levels (182.4 mg/100 mL), likely due to enhanced release from cell structures and reduced oxidation, as reported by Rawson et al., (2011). The hypothesis that ultrasound aids in the extraction of phenolics and antioxidants is supported by the fact that antioxidant activity increased under optimal conditions (93.20%), with amplitude and its quadratic effect being most significant (Tiwari et al., 2009). Colour value was also affected mainly by amplitude (F = 10.01), showing slight increases at higher amplitudes due to pigment degradation. Optimization using a desirability function yielded ideal conditions at 83.88% amplitude and 14.25 minutes, producing amla juice with minimal microbial load (1.495 log CFU/mL), high ascorbic acid and antioxidant levels and acceptable colour, with an overall desirability of 0.611, further increase in the amplitude increases microbial quality but decrease in the nutrients were observed due to production of free radicle at higher amplitudes. These results demonstrate that ultrasonic is a successful non-thermal juice processing technique that improves nutritional quality and microbiological safety without appreciably degrading sensory qualities (Adekunte et al., 2010). Table 1 and 2 display the model coefficients and ANOVA data of the various qualitative parameters of ultra-sound processed amla juice, as well as the restrictions and constraints used to optimize the ultra-sound processing.




 
Microbial quality, ascorbic acid, antioxidant activity and colour value
 
Ultrasound assisted processing exerted a pronounced effect on the microbial quality of amla juice, with microbial load decreasing significantly as ultrasonic amplitude and processing time increased. The control sample exhibited an initial microbial count of 2.36 log CFU/mL, whereas the optimized treatment resulted in a reduced count of 1.495 log CFU/mL. Notably, complete microbial inactivation (0 log CFU/mL) was achieved at 95% amplitude for 15 min, highlighting the strong antimicrobial efficacy of high-intensity ultrasonication. According to FSSAI standards, the permissible limit for total plate count in fruit juices is ≤5.0 x 10⁴ CFU/mL (≈4.7 log CFU/mL), with the absence of pathogenic microorganisms. All ultrasound-treated samples in the present study remained well within these regulatory limits, thereby confirming the microbiological safety of the processed juice. The observed microbial reduction can be attributed to acoustic cavitation, which generates localized high temperatures and pressures, leading to cell membrane disruption, intracellular leakage and eventual microbial cell death (Chemat et al., 2011). The enhanced lethality observed at higher amplitudes and extended processing times reflects increased cavitational intensity and energy input, which collectively amplify mechanical and physicochemical stress on microbial cells. Ascorbic acid content exhibited a non-linear response to ultrasound treatment. Moderate ultrasonication (85% amplitude for 10 min) resulted in a slight increase in ascorbic acid content (182.35 mg), likely due to improved extractability arising from ultrasound-induced cell wall disruption. However, further increases in amplitude and processing time led to a decline in ascorbic acid levels, particularly at 95% amplitude. Acoustic cavitation, which produces localized high temperatures and pressures that cause cell membrane breakdown, intracellular leaking and ultimately microbial cell death, is responsible for the observed microbial decline (Adekunte et al., 2010). These findings indicate that while ultrasound can enhance nutrient release at moderate intensities, excessive energy input may negatively affect bioactive compounds.
       
Antioxidant activity showed a similar trend, with the highest value (93.4%) recorded at 75% amplitude for 15 min. The initial enhancement in antioxidant activity is likely associated with the release of bound phenolic and antioxidant compounds from disrupted cellular matrices. Comparable observations have been reported for kiwifruit juice, where high-intensity ultrasound significantly enhanced antioxidant capacity (Wang et al., 2019). However, a slight decline at higher amplitudes and prolonged treatment durations suggests partial degradation of labile antioxidant constituents, as previously reported by Ghafoor et al., (2009). Overall, antioxidant activity remained relatively stable across treatments, indicating that ultrasound processing effectively preserves antioxidant potential within an optimal processing window.
       
Colour variation (ΔE) increased marginally with increasing amplitude and processing time, ranging from 2.61 to 3.42, yet remained within acceptable sensory limits. The observed colour changes may be attributed to enhanced pigment release and mild browning reactions induced by prolonged sonication. The highest colour difference (ΔE = 3.42) was observed at 95% amplitude for 15 min, suggesting that excessive processing intensity may influence visual appearance. Nevertheless, the magnitude of colour change was minimal and unlikely to negatively impact consumer acceptance, consistent with previous reports on cantaloupe melon, peach and orange juices subjected to ultrasound processing (Fonteles et al., 2012; Rojas et al., 2016; Valero et al., 2007). Similar subtle colour modifications (TCD < 2.5) have also been reported in ultrasound-treated red grape juice, indicating negligible visual degradation (Tiwari et al., 2010). The response surface plots illustrating the combined effects of amplitude and time on these quality attributes are presented in Fig 1.


 
pH, TSS and titratable acidity
 
Ultrasound treatment did not significantly affect the pH of amla juice, with values remaining within a narrow range (2.91-2.97) across all treatments. This stability implies that ultrasonication does not cause significant acid-base reactions in the juice matrix or change the concentration of hydrogen ions. On the other hand, titratable acidity increased slightly but steadily as amplitude and processing time increased (from 2.02% to 2.126% citric acid equivalents). This could be explained by the increased release of organic acids from damaged plant tissues during ultrasonication (Rawson et al., 2011).
       
Total soluble solids (TSS) exhibited a marginal increase from 7.4 to 7.5 °Brix following ultrasound treatment.  The reason was cavitation-induced structural disruption which causes release of intracellular soluble solids and degradation of complex polysaccharides (Kentish and Feng, 2014). The slight alterations show increased solute availability without sacrificing juice quality.
 
Total phenolic content and flavonoids (TPC)
 
Ultrasound processing significantly enhanced TPC of amla juice. The control sample contained 372.65 mg GAE/100 g, whereas the highest TPC (533.34 mg GAE/100 g) was observed at 95% amplitude for 15 min. This significant increase is explained by the rupture of cell walls and membranes caused by cavitation, which makes it easier for bound phenolic chemicals to be released into the juice matrix (Rawson et al., 2011). A similar increase in TPC was observed in finger millet due to release of bound polyphenols (Adoni et al., 2025). The antioxidant and health-promoting qualities of amla juice are mostly attributed by the phenolic chemicals, whose increased availability highlights the practical benefits of ultrasonic processing. Pineapple juice treated with high-intensity ultrasound has been shown to exhibit comparable increases in phenolic content (Costa et al., 2013).
       
Flavonoid content exhibited a comparable increasing trend. The control sample contained 310.47 mg QE/100 g, which increased to 393.28 mg QE/100 g at 95% amplitude for 15 min. The non-thermal nature of ultrasound plays a critical role in preserving heat-sensitive flavonoids while simultaneously improving their extractability. Treatments at 85% amplitude for 10-15 min also yielded significantly high flavonoid levels (369.95-379.95 mg QE/100 g), indicating this range as optimal for maximizing bioactive retention. A similar increase in flavonoid extraction occurred when lotus seeds were subjected to ultrasound treatment (Long et al., 2020). These findings align with reports on kiwifruit juice, where high-intensity ultrasound enhanced phenolics, flavonoids and individual antioxidant compounds with increasing treatment time (Wang et al., 2019).
       
Overall, the results presented in Table 3 demonstrate that ultrasound-assisted processing effectively enhances the functional quality of amla juice by increasing phenolic and flavonoid contents, which are closely associated with antioxidant capacity, disease prevention and consumer appeal. The study highlights the potential of optimized ultrasound parameters as a promising non-thermal technology for producing nutritionally superior and microbiologically safe amla juice.

The present study demonstrated that ultrasound processing effectively improves the biochemical, microbial and sensory qualities of amla (Phyllanthus emblica. L) juice. Optimized conditions, particularly treatment at 84% amplitude and 14.25 minutes, resulted in significant microbial inactivation while promoting the retention of ascorbic acid, antioxidant activity and bioactive compounds such as phenolics and flavonoids. Colour stability was also well maintained under these conditions. These results highlight the potential of ultrasound-assisted techniques as a green and efficient alternative to conventional thermal processing for the preservation of fruit juices, with minimal nutritional and sensory losses. Future research should evaluate cost benefit trade-offs associated with different ultrasound treatments and scale-up challenges related to acoustic field uniformity, equipment design and continuous-flow operation should be systematically investigated. Pilot-scale studies are necessary to determine whether the optimized laboratory conditions can be reliably translated to industrial systems without compromising product quality or process efficiency. Integration of ultrasound with existing thermal or non-thermal preservation techniques (e.g., mild heat or pulsed electric fields) may also be explored to reduce processing intensity while maintaining safety and quality.

The present study was supported by Anand Agricultural University.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.