Thermal-spring bacterium yields heat-stable amylase that thins porridge by 90%

A thermophilic bacterium recovered from a hot spring in western India has yielded a heat-stable alpha-amylase that, after statistical optimization of its production, cut the viscosity of wheat porridge by roughly 90 percent within minutes. The enzyme, produced by Laceyella sacchari strain THS-B4, was isolated from the Tuva thermal spring in Gujarat and characterized in a laboratory study published in the Springer journal Discover Bacteria. The work combines bioprocess engineering with a nutritional application: the same enzyme that thrives near boiling temperatures was tested as a thinning agent for weaning foods, where high viscosity is a well-known barrier to delivering enough energy to infants.

The research team, led by Hiral H. Padia and including Krushali S. Jain, Vaishnavi C. Goswami and Devayani R. Tipre, screened water samples collected from the Tuva hot spring and grew them on starch agar at 50 degrees Celsius. Forty-two bacterial isolates showed clear starch-hydrolysis halos when the plates were flooded with iodine, and one isolate, designated B4, displayed the highest amylolytic index at 2.8 plus or minus 0.1. Molecular identification based on a 931-base-pair partial 16S rRNA gene sequence showed 100 percent similarity with Laceyella sacchari strain P43, and phylogenetic analysis using the neighbour-joining method placed the new strain firmly within the L. sacchari lineage with a bootstrap value of 94 percent from 1000 replicates. The sequence was deposited in GenBank under accession number PQ135078. The organism proved to be a Gram-positive, spore-forming thermoactinomycete that grew optimally at 50 to 55 degrees Celsius and showed no growth at mesophilic temperatures, confirming its strictly thermophilic character. The authors caution that 16S rRNA sequencing resolves genus-level identity reliably but has limited power to distinguish closely related species and strains, so the taxonomic assignment should be considered preliminary until whole-genome sequencing is performed.

With the producer organism in hand, the team turned to statistical experimental design to raise enzyme yields. A Plackett-Burman design tested eight medium variables at two levels each, including starch, yeast extract, peptone, dipotassium hydrogen phosphate, sodium chloride, magnesium sulphate, tryptone and medium pH, with three dummy variables included to estimate experimental error. The screening identified starch concentration, NaCl, tryptone and pH as the most significant factors influencing amylase production, each with a p-value below 0.001. Starch exerted a significant positive effect on yield, consistent with its role as both carbon source and inducer of amylase synthesis, while NaCl, tryptone and pH showed significant negative effects within the tested ranges, indicating that the high levels used in screening exceeded the strain’s physiological optimum. The first-order regression model from this stage achieved an R-squared of 0.9478 and an adjusted R-squared of 0.9269, with a model F-value of 45.38.

The second optimization stage used a Central Composite Design within Response Surface Methodology, with 20 experimental runs conducted in triplicate to refine three variables: starch concentration from 1.5 to 2.5 grams percent, NaCl from 0.5 to 1.0 grams percent, and medium pH from 6.5 to 7.5. Notably, tryptone was excluded from this stage despite its statistical significance, because adding a third complex nitrogen source on top of yeast extract and peptone would raise raw material costs without proportional yield gains; validation experiments showed that omitting tryptone while retaining peptone maintained high specific activity at roughly 60 percent lower material cost. The resulting second-order polynomial model captured 99.48 percent of the variation in enzyme activity, with an adjusted R-squared of 0.9902 and a predicted R-squared of 0.9658 in close agreement. Analysis of variance confirmed highly significant interactions between starch and pH and between NaCl and pH, each with p-values below 0.0001, while the starch-NaCl interaction was significant but weaker at p equal to 0.0010. Response surface plots pointed to an optimum at 2 grams percent starch, 0.75 grams percent NaCl and pH 7.

Validation runs at those conditions, carried out in triplicate at 50 degrees Celsius for 48 hours, produced an experimental amylase yield of 4.24 plus or minus 0.08 units per millilitre against a model prediction of 4.30 units per millilitre, a relative prediction error of just 1.39 percent. That yield corresponds to a 3.8-fold enhancement over the unoptimized baseline, achieved purely through medium tuning rather than genetic manipulation. The quadratic terms in the model all carried negative coefficients, indicating a parabolic response and a true optimum inside the experimental range rather than a boundary effect. The authors interpret the positive starch-NaCl interaction as evidence that higher substrate loads require appropriate osmotic regulation for efficient enzyme secretion, and they suggest NaCl may also support ion transport, protein secretion and enzyme stability under thermophilic conditions, though these mechanistic interpretations remain hypotheses in the absence of structural data.

Purification proceeded through sequential ammonium sulphate precipitation at 50 and 80 percent saturation, gel filtration on Bio-Gel P100, and ion-exchange chromatography on DEAE-cellulose. The final preparation achieved 33.7-fold purification with a 12.2 percent yield and a specific activity of 33.7 units per milligram. SDS-PAGE revealed a single band at approximately 62 kilodaltons, confirmed by zymographic analysis in which enzymatic starch degradation appeared as clear bands against a darkly stained background. The authors note that the modest recovery reflects the multi-step laboratory protocol; for industrial use, simpler downstream approaches such as ultrafiltration, membrane concentration or spray drying would be more appropriate and could potentially achieve substantially higher recovery while maintaining activity. For comparison, they cite an amylase from L. sacchari TSI-2 purified 55-fold with 12 percent recovery, and Bacillus amyloliquefaciens TSWK1-1 purified 13-fold with 46 percent yield.

Biochemical characterization showed an enzyme well suited to harsh processing environments. Activity was measured from 30 to 90 degrees Celsius, with maximum activity at 80 degrees and 74 percent of that maximum retained at 90 degrees. The enzyme kept roughly 70 to 95 percent of its activity during incubation at 40 to 50 degrees Celsius, held nearly 50 percent of its initial activity after two hours at 60 degrees, and declined sharply after 60 minutes at 80 degrees. Across the pH scale, activity was assessed from 4 to 9, with more than 60 percent of activity retained between pH 5 and 9 and maximum activity at pH 7. Stability testing over 210 minutes showed only marginal losses from pH 6 to 9, with roughly 45 to 60 percent residual activity across that range. Metal ion profiling revealed enhanced activity in the presence of calcium, magnesium, sodium and ferrous ions and inhibition by copper, while the chelator EDTA inhibited activity at 10 millimolar. Most strikingly, the enzyme retained approximately 99 percent residual activity in the anionic surfactant SDS at concentrations up to 0.5 percent, with dose-response tests at 0.1, 0.2 and 0.5 grams percent SDS yielding 101.4, 99.8 and 99.1 percent activity relative to untreated controls. Because SDS typically inactivates enzymes, this tolerance is rare among bacterial amylases and suggests the protein resists denaturation, although the molecular basis remains unexplained since no sequence or structural analyses were performed.

Kinetic analysis used eleven soluble starch concentrations from 0.1 to 3.0 milligrams per millilitre, each assayed in triplicate. Nonlinear regression against the Michaelis-Menten model produced a Km of 0.753 milligrams per millilitre with a 95 percent confidence interval of 0.627 to 0.904, and a Vmax of 1.603 micromoles per minute per millilitre with a confidence interval of 1.505 to 1.714. The fit achieved an R-squared of 0.9686, and a Lineweaver-Burk double-reciprocal plot corroborated the estimates. The authors place these values alongside reported constants for other thermophilic amylases, including Anoxybacillus rupiensis TS-4, describing the substrate affinity and catalytic performance as falling within a similar range.

The nutritional application test compared the THS-B4 enzyme head-to-head with a commercial Bacillus amylase on freshly cooked wheat porridge at 50 to 60 degrees Celsius with a five-minute incubation. In 20 percent wheat porridge, the THS-B4 treatment reduced viscosity from 15277.0 plus or minus 25.5 centipoise to 1528.3 plus or minus 32.2 centipoise, a 90.15 percent reduction, while the commercial enzyme achieved 1467.3 plus or minus 17.0 centipoise, a 90.50 percent reduction. In 25 percent porridge, THS-B4 cut viscosity from 22348.3 plus or minus 32.6 to 2616.0 plus or minus 11.5 centipoise, an 88.34 percent reduction, versus 2479.0 plus or minus 17.6 centipoise and 88.90 percent for the commercial preparation. Both treatments converted spoonable, highly viscous porridges into free-flowing consistencies. Free sugar content rose from 4.68 to 6.84 milligrams per millilitre in the 20 percent porridge and from 6.23 to 7.32 in the 25 percent porridge, indicating efficient starch hydrolysis into smaller, more soluble carbohydrates. The measurements used three independent porridge preparations with triplicate viscosity and sugar readings, and untreated controls were run under identical conditions.

The practical significance lies in weaning foods, where starch gelatinisation during cooking produces gels so thick that infants cannot consume enough volume to meet energy needs. Reducing viscosity allows higher solid content without increasing thickness, raising energy density, and the released sugars may also add mild sweetness that improves palatability. The authors note that amylase-rich flour from germinated grains has traditionally served this purpose but its preparation is labour-intensive and demands strict control of germination conditions, whereas direct enzyme addition offers rapid, controlled thinning at low dosage. The team emphasizes that this is, to their knowledge, the first report of a Laceyella alpha-amylase applied to cereal porridge thinning. They also stress the limits of the work: the study was conducted at laboratory scale, stability under industrial conditions such as detergent formulations, prolonged thermal processing and varying salinity was not evaluated, and pilot-scale validation, whole-genome sequencing and structural characterization remain future steps. Within those boundaries, the combined profile of thermostability, surfactant tolerance and porridge-thinning performance comparable to a commercial enzyme positions THS-B4 as a candidate worth further development for both bioprocess and nutritional applications.

Subject of Research: Statistical optimization of a thermostable alpha-amylase from the thermophilic bacterium Laceyella sacchari THS-B4 and its use in thinning cereal porridge

Article Title: Statistical optimization of thermostable alpha amylase production by Laceyella sacchari THS-B4 and its application in cereal porridge thinning

Article References: Padia, H. H., Jain, K. S., Goswami, V. C., & Tipre, D. R. (2026). Statistical optimization of thermostable alpha amylase production by Laceyella sacchari THS-B4 and its application in cereal porridge thinning. Discover Bacteria, 3(1), Article 21. https://doi.org/10.1007/s44351-026-00059-5

Image Credits: AI Generated

DOI: 10.1007/s44351-026-00059-5

Keywords: Laceyella sacchari, alpha-amylase, thermophilic bacteria, hot spring, Plackett-Burman design, response surface methodology, enzyme purification, porridge thinning, weaning foods, viscosity reduction, SDS stability, Michaelis-Menten kinetics