Fresh stem mustard registered a hardness of 163.70 newtons and a chewiness of 136.35 millijoules, measured with a texture analyzer using a 2 millimeter cylindrical probe at 55 percent compression. After the first pickling stage, in which 3 percent salt is added for about five days before pressing, hardness had already plunged to 26.83 newtons. It stayed near that level through the second, 10-day stage at 5 percent salt, at 25.03 newtons, then dropped further to 10.47 newtons after the third and longest stage, a 90-day fermentation at 8 percent salt that both develops flavor and, at its high salt concentration, inhibits microbial growth to allow extended storage.

Chewiness followed a similar trajectory, falling to 15.02 millijoules by the end. The most dramatic single loss occurred in that earliest stage, which the researchers identify as the critical window for intervention. Physicochemical measurements supplied the environmental backdrop for this decline.

Moisture content fell from 93.94 grams per 100 grams in fresh samples to 79.66 grams per 100 grams at the end of pickling, a consequence the authors attribute to a salting-out effect: as salt penetrates the tissue, damaged cell walls release water and the tissue dehydrates. Total acid, the hallmark of lactic acid fermentation, climbed from 1.32 grams per kilogram to 5.12 grams per kilogram as microbial growth and metabolism accelerated in the later stages, converting more sugars into organic acids. Nitrite, a critical safety parameter for pickled vegetables, declined steadily and ended at just 1.83 milligrams per kilogram, far below the 20 milligrams per kilogram benchmark cited in the study, reflecting the denitrifying and nitrate-reducing activity of fermentative microorganisms reported in earlier work on Zhacai and cowpea pickles.

In fresh tubers, the carbonate-soluble fraction dominated at 102.85 milligrams per gram, with chelate-soluble pectin at 49.15 and only 7.65 milligrams per gram of the water-soluble form, a pattern indicating an intact, well-crosslinked wall. As pickling proceeded, the alcohol-insoluble residue and the insoluble pectin fractions declined continuously while water-soluble pectin rose, a chemical signature of cell wall disassembly. Carbonate-soluble pectin is covalently anchored into the wall's polysaccharide network, so its loss thins and weakens the wall and loosens cell connections.

Chelate-soluble pectin, held in the middle lamella by calcium bridges, glues neighboring cells together; its decline loosens intercellular adhesion. The researchers suggest that salt ions in the brine disrupt those ionic bonds while the enzyme pectin methylesterase promotes conversion of the chelate-soluble form into the weakly bound, water-soluble type, which attaches to the wall only through non-covalent, non-ionic interactions. The enzyme measurements explained how this conversion was catalyzed.

Activities of pectin methylesterase, polygalacturonase and beta-galactosidase all followed the same pattern: a sharp early rise followed by a decline across the later two stages. The authors propose that the pressing step physically disrupts cell architecture, releasing enzymes from their compartments, while the increasing salt concentration later creates osmotic stress that damages catalytic sites and compromises enzymatic function, a phenomenon also observed in salt-stressed tomato. Each enzyme has a distinct role.

Pectin methylesterase strips methyl-ester bonds from galacturonic acid residues, lowering the degree of esterification and making pectin vulnerable to attack. Polygalacturonase then cleaves the exposed galacturonic acid backbone, reducing cell rigidity; its early peak in the first-stage sample coincided with a supply of low-esterified substrate created by the methylesterase. Beta-galactosidase trims galactosyl side chains, weakening interactions between pectin molecules.

The team also notes that microbes such as Providencia vermicola and Galactomyces geotrichum, reported in earlier studies, can secrete their own plant cell wall-degrading enzymes during fermentation and contribute to vegetable softening. Cellulose, the structural polysaccharide that forms microfibers and supports tissue rigidity, degraded in parallel. Cellulose content was highest in fresh samples and fell steadily as pickling advanced, and cellulase activity peaked at the end of the first stage before declining.

The authors link this early cellulase boost to the initial salt concentration and its later suppression to the accumulating acidity of the fermenting brine, a pattern previously seen in dry-salted spaghetti squash and fermented chopped peppers. Similar cellulose degradation has been documented in pickled radish, where both microbial exogenous enzymes and liberated endogenous enzymes appear to contribute under saline conditions. Correlation and clustering analyses drew the threads together.

Hierarchical clustering showed the first and second pickling stages grouping together with the third stage joining them, while fresh samples formed an entirely separate branch, evidence of progressively divergent cell wall metabolism as pickling proceeded. The authors attribute this divergence to the relatively short duration of the first two stages, where quality changes were driven mainly by salt penetration and wall-degrading enzymes, whereas the third stage is characterized predominantly by lactic acid bacteria fermentation. Pickling time correlated negatively with moisture, carbonate-soluble pectin, chelate-soluble pectin and cellulose, and positively with water-soluble pectin and wall-degrading enzyme activities.

Hardness and chewiness correlated positively with the insoluble wall fractions and moisture but negatively with water-soluble pectin, indicating that cell wall composition and moisture content play a dominant role in determining Zhacai texture. Intriguingly, no significant correlation emerged between the texture parameters and cell wall-degrading enzyme activity directly, a result also reported for white kiwifruit. The authors interpret this as evidence that softening is a cumulative, multi-factor effect: enzyme activities rose and then fell while hardness declined continuously, and the synergy among different endogenous enzymes weakens the relationship between any single enzyme and the texture indicators, with high salt further suppressing enzymatic contribution in later stages.

Enzyme activities did correlate significantly with the cell wall fractions, with beta-galactosidase and polygalacturonase showing the most prominent associations, and total acid was negatively correlated with the activities of those two enzymes, suggesting that accumulating organic acids help modulate the rate of wall disassembly in later pickling. Fourier-transform infrared spectroscopy added a molecular view. Spectra of the pectin fractions showed characteristic polysaccharide bands, and the degree of esterification, calculated from the ratio of esterified to total carboxyl band areas, fell continuously through pickling.

In the water-soluble fraction it dropped from 63.03 percent in fresh tissue to 39.40 percent in the final product, and in the chelate-soluble fraction from 55.45 percent to 33.31 percent. This progressive de-esterification triggers the breakdown of water-insoluble pectin into water-soluble components, loosening the wall. Peak shifts near 1012 per centimeter indicated changes in pyranoside bonds during pickling.

Overall, however, the authors report no dramatic changes in pectin components or cellulose structure across the process, with only several pectin-associated functional groups, such as the C–O–O band of water-soluble pectin near 1407 per centimeter, changing at the late stage, implying alterations in pectin monosaccharide composition. Transmission electron microscopy made the damage visible at the subcellular scale. Fresh cells showed an intact, continuous wall of uniform thickness, a smooth membrane pressed against the wall, orderly mitochondria with dense cristae and a dominant central vacuole.

By the first pickling stage, walls had become unevenly thickened and folded in places, the middle lamella had loosened and delaminated, and plasmolysis had ruptured membranes, scattering fragmented organelles through the cytoplasm. These defects worsened through the second and third stages, culminating in severely distorted walls with heterogeneous thickness and pervasive organelle debris. The authors also observed occasional wall thickening, which they attribute to the pressing step: mechanical pressure prompting cell wall components to retain cell fluid can trigger swelling-induced gelation, as previously described in pumpkin cubes.

Because the first pickling stage is where wall-degrading enzymes surge, the authors suggest that inhibiting enzyme activity at this early point offers the best chance of preserving texture, and that alternative, non-mechanical dehydration approaches could reduce the cell wall damage caused by pressing. They caution that their experimental design could not quantitatively separate the contributions of mechanical force and enzymatic hydrolysis, and that exogenous pectinases and cellulases from halophilic lactic acid bacteria during late fermentation may also participate in polysaccharide breakdown. The study examined a single traditional production process, with correlations rather than controlled single-factor experiments, so the interplay among salt stress, enzymatic hydrolysis, microbial metabolism and wall restructuring will require further disentangling.

Subject of Research: Mechanisms of texture softening in Fuling Zhacai during the traditional three-stage pickling process

Article Title: The effect of key physicochemical properties, cell wall polysaccharides, and cell microstructure on the texture of Fuling Zhacai during pickling process

Article References: Zheng, Q., Zhang, Z., Rao, M., Pan, Y., Song, J., & Gao, X. (2026). The effect of key physicochemical properties, cell wall polysaccharides, and cell microstructure on the texture of Fuling Zhacai during pickling process. Food Chemistry: X, 40, Article 104630. https://doi.org/10.1016/j.fochx.2026.104630

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104630

Keywords: Fuling Zhacai, pickling, texture softening, pectin, cellulose, cell wall-degrading enzymes, fermentation, transmission electron microscopy, FT-IR, food science, stem mustard, salt

Indian borage, known scientifically as Coleus amboinicus and sometimes called Mexican mint or country borage, is a fleshy-leaved herb in the mint family whose aroma comes from a dense payload of volatile compounds. Neem, Azadirachta indica, is a tree revered across South Asia for centuries of medicinal use, and its leaves contain a complex mixture of limonoids, flavonoids and other defensive chemicals. Both plants have long folk histories, but the research team set out to address a more modern problem: the limited availability of sustainable and efficient extraction methods that reliably deliver bioactive compounds from these leaves. Their approach was to characterise the extracts not just for the headline activities, but for the physicochemical fingerprints that explain them, including colour, chlorophyll content, functional groups and volatile profiles.

The experimental design hinged on a single variable with outsized consequences: solvent polarity. The researchers prepared ethanolic extracts of both leaves using two concentrations, 50 percent and 95 percent ethanol, the remainder being water. This seemingly small difference in the water-alcohol ratio changes which molecules the solvent can dissolve. Water-rich mixtures favour hydrophilic compounds such as phenolic acids and polar flavonoids, while nearly pure ethanol preferentially pulls out hydrophobic constituents such as terpenoids and lipophilic phenolics. By running both conditions side by side for both plants, the team could directly attribute differences in biological activity to the chemistry that each solvent mobilised, rather than to the plant species alone.

To measure antioxidant power, the team used the DPPH assay, a widely adopted test in which a stable purple free radical is decolourised when an antioxidant donates a hydrogen atom or electron. The results were striking for the 50 percent ethanol extracts, which achieved DPPH radical scavenging activity between 87 and 89 percent. That level of radical quenching indicates that the half-strength ethanol was highly effective at extracting hydrophilic antioxidants, the water-soluble phenolic compounds that plants deploy against oxidative stress. For food formulators, such extracts could serve as natural alternatives to synthetic antioxidants, which have faced increasing consumer scrutiny, in products where preventing lipid oxidation and colour degradation is the priority.

The antimicrobial story inverted that picture. When the 95 percent ethanol extracts were tested against common foodborne pathogens, they produced inhibition zones ranging from 1.06 to 1.52 centimetres, a measure of how far the antibacterial effect spread from a test well or disk in an agar plate assay. The team attributed this stronger antimicrobial performance to the extraction of hydrophobic bioactive compounds, which can integrate into and disrupt bacterial cell membranes. Pathogens of this kind include organisms responsible for food spoilage and foodborne illness, so an extract that suppresses their growth could extend shelf life or improve safety. The practical implication is that a manufacturer seeking an antimicrobial ingredient should reach for the stronger ethanol, while one seeking an antioxidant should choose the weaker one.

Beyond activity assays, the study built a detailed physicochemical portrait of each extract. Colour was quantified with a Chromameter CR-400, an instrument that converts visual appearance into objective numerical values, an important step for any ingredient destined for food or cosmetics where appearance drives consumer acceptance. Chlorophyll-a, chlorophyll-b and total chlorophyll were measured spectrophotometrically, exploiting the characteristic wavelengths at which these pigments absorb light. Chlorophyll is not merely a marker of plant material; its degradation products and associated compounds such as phytol have drawn scientific interest in their own right, and pigment content influences both the colour stability and the potential bioactivity of a leaf extract during storage and processing.

Functional group analysis was carried out with Fourier-transform infrared spectroscopy, or FT-IR, a technique that shines broadband infrared light through a sample and records which frequencies are absorbed. Each absorption band corresponds to a specific molecular bond, such as the stretches of hydroxyl groups in phenolics, carbonyl groups in acids and esters, or carbon-hydrogen bonds in lipids and terpenes. By comparing the spectra of the 50 percent and 95 percent ethanol extracts, the researchers could confirm at the molecular level that the two solvents had indeed pulled out chemically distinct suites of compounds, providing a mechanistic underpinning for the divergent antioxidant and antimicrobial results.

The most granular layer of characterisation came from gas chromatography-mass spectrometry, or GC-MS, which separates volatile compounds in a heated column and then identifies each one by the characteristic fragmentation pattern it produces when bombarded with electrons. This technique is ideally suited to plants like Indian borage, whose aromatic identity is defined by volatile terpenes, and to neem, whose leaf chemistry includes an array of volatile and semi-volatile constituents. The volatile profiles documented in the study help explain both the traditional sensory appeal of these plants and the biological activities observed, since many terpenoids are known to contribute to both antioxidant defences and membrane-disrupting antimicrobial action.

The broader significance of the work lies in its framing of solvent polarity and processing methods as design levers rather than fixed choices. Instead of asking simply whether a plant extract is active, the study demonstrates that the same leaf can yield two functionally different ingredients depending on a single processing parameter that is cheap and easy to control industrially. That flexibility is valuable for the growing market for clean-label preservatives and bioactive ingredients, where producers want predictable performance. It also supports sustainability goals: leaves of widely cultivated, fast-growing plants can be valorised as sources of high-value compounds, reducing reliance on synthetic antioxidants and antimicrobials derived from petrochemical feedstocks.

The authors, whose collaboration spanned the Faculty of Applied Sciences and Faculty of Health Sciences at Universiti Teknologi MARA, Auckland University of Technology in New Zealand, and Universitas Brawijaya in Indonesia, emphasise the potential of Coleus amboinicus and Azadirachta indica as sustainable sources of antioxidants and antimicrobials for applications spanning food, pharmaceuticals and cosmetic products. As with any laboratory study, the path from inhibition zones and spectrophotometer readings to commercial products will require further work on safety, dosage, stability and sensory impact in real formulations. But the core message is already clear and actionable: when it comes to extracting useful chemistry from these two familiar leaves, the solvent is not just a medium, it is the recipe.

Subject of Research: Solvent-dependent antioxidant and antimicrobial activities of ethanolic leaf extracts from Indian borage and neem

Article Title: Antioxidant and antimicrobial activities of Indian borage leaves (Coleus amboinicus, Lamiaceae) and neem leaves (Azadirachta indica, Meliaceae) ethanolic extracts

Article References: Khairunnisa-Yusra, A. R., Wan-Razarinah, W. A. R., Aida, F. M. N. A., Dasiman, R., Hamid, N., Huda, N., Murtini, E. S., & Raseetha, S. (2026). Antioxidant and antimicrobial activities of Indian borage leaves (Coleus amboinicus, Lamiaceae) and neem leaves (Azadirachta indica, Meliaceae) ethanolic extracts. Plant Biosystems, 160(5), Article 277. https://doi.org/10.1007/s44473-026-00272-9

Image Credits: AI Generated

DOI: 10.1007/s44473-026-00272-9

Keywords: Indian borage, neem, Coleus amboinicus, Azadirachta indica, ethanolic extraction, antioxidant activity, antimicrobial activity, DPPH assay, GC-MS, FT-IR, chlorophyll, natural preservatives