Enhancing the probiotic, sensory, and antioxidant properties of Polygonatum sibiricum Red. and Crataegus pinnatifida Bunge through lactic acid fermentation for functional food development

Abstract

This study explores the use of Polygonatum sibiricum Red. and Crataegus pinnatifida Bunge as edible medicine homology ingredients through lactic acid fermentation to enhance probiotic content, sensory quality, and antioxidant properties. Using a mixed culture of Lactobacillus plantarum AS1.2437 and Lactobacillus casei CICC21019, the fermentation significantly increased lactic acid bacteria counts, improved taste, and boosted antioxidant activities, particularly in reducing Fe³⁺ and scavenging 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) free radicals (p < .05). During fermentation, changes in reducing sugars, total acids, superoxide dismutase (SOD) activity, and amino acid compositions were monitored. Results showed significant increases in total phenols, flavonoids, SOD activity, and polysaccharide content (p < .05). Essential amino acids increased by 4.86%, and therapeutic amino acids rose from 50.88% to 53.59%. The Boltzmann model effectively captured these changes, achieving a high coefficient of determination (R² > 0.99), indicating strong predictive accuracy. Key organic acids like lactic, acetic, and tartaric acids contributed to enhanced antioxidant activity. This research provides a comprehensive understanding of the physicochemical, medicinal, and antioxidant properties of the fermented Polygonatum sibiricum Red. and C. pinnatifida Bunge (PsR.–CpB.) enzyme, establishing a theoretical foundation for developing functional foods aligned with the edible medicine homology concept.

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Introduction

With the improvement of living standards and the enhancement of health awareness, the concept of edible medicinal homology has gradually gained attention (Shang et al., 2021). Edible medicinal homology refers to the use of plant and animal materials that can serve both as food and medicine, which are widely utilised in traditional Chinese medicine. Among these, Polygonatum sibiricum Red. and Crataegus pinnatifida Bunge (Wei et al., 2024), as traditional medicinal plants in China, have significant applications in pharmacology and are rich in various nutritional components. The combination of these two herbs not only enhances the flavour of the products but also increases their functionality, providing a solid foundation for the development of health foods. Lactic acid fermentation is a classical food fermentation technique, widely applied in the production of dairy products, fermented vegetables, and other foodstuffs (Jabłońska-Ryś et al., 2019). This process can enhance the safety, nutritional value, and flavour of foods while generating various bioactive compounds that contribute to improving human health. Lactic acid fermentation is particularly beneficial due to its ability to promote beneficial gut microbiota, enhance the bioavailability of nutrients, and produce antimicrobial compounds, which altogether improve the overall health benefits of the fermented product. The process also contributes to the preservation and enhancement of the flavours of food products, making it a versatile choice for developing functional foods (Yang et al., 2024).

In recent years, researchers have identified that Polygonatum sibiricum Red. and C. pinnatifida Bunge, as sources of edible medicinal homology, play a significant role in regulating hyperlipidaemia, improving gastrointestinal function, and addressing sub-health conditions (Li et al., 2022a, 2023a). For instance, it indicated that polysaccharides and flavonoids present in Polygonatum sibiricum Red. significantly had improved metabolic syndrome (Lai et al., 2024). Similarly, active compounds in C. pinnatifida Bunge facilitated digestion, enhanced gastrointestinal health, and exhibited promising lipid-lowering effects (Jing et al., 2023). To optimise the utilisation of these resources, the preparation of edible plant enzymes has emerged as a research focus. Fermenting these edible medicinal homology plants with probiotics not only enhances the proliferation of beneficial bacteria but also maximises the retention of their inherent nutritional components while introducing new functional factors (Dai et al., 2020). These fermented enzymes are regarded as products with nutritional, health, and dietary therapeutic benefits, presenting substantial market potential (Fang et al., 2020). Relevant studies have confirmed that the beneficial components and bioactive substances in fermented products are significantly increased (Jiang et al., 2021; Li et al., 2022b), further expanding their application areas. In this study, we focused on the fermentation of Polygonatum sibiricum Red. and C. pinnatifida Bunge using appropriate lactic acid bacteria to develop a novel enzyme rich in flavour and functionality. The kinetic models for the consumption of reducing sugar substrates, total acid production, and the growth of lactic acid bacteria during the fermentation were investigated. Additionally, the physicochemical properties, microbial composition, essential and medicinal amino acids, organic acids, and antioxidant activity of the products before and after fermentation were analysed. The goal is to provide a theoretical basis for the precise preparation and quality analysis of lactic acid bacteria-fermented Polygonatum sibiricum Red. and C. pinnatifida Bunge enzyme (PsR.–CpB. enzyme). This research not only aids in a deeper understanding of the biochemical changes occurring during fermentation but also contributes to the optimization of fermentation processes, thereby improving the quality and functionality of the final enzyme products.

Materials and methods

Material, standards, and reagents

Polygonatum sibiricum Red., identified as a species of the Liliaceae family, was sourced from Henan Lianyuan Biotechnology Co., Ltd. Dried C. pinnatifida Bunge fruit was purchased from a supermarket in Tongpai County, Nanyang City, Henan Province. Brown sugar was procured from the local market. Lactobacillus plantarum AS1.2437 and Lactobacillus casei CICC21019 were preserved in glycerol and obtained from the Beijing Biological Resource Center. Pectinase (500 U/mg), cellulase (50 U/mg), and standard rutin were acquired from Shanghai Yuanye Biotechnology Co., Ltd. DeMan, Rogosa and Sharpe (MRS) broth and MRS agar were obtained from Beijing Aoboxin Biotechnology Co., Ltd. Folin–Ciocalteu’s phenol reagent was sourced from Shanghai Macklin Biochemical Co., Ltd. Analytical grade sodium nitrite was purchased from Tianjin Dengke Reagent Co., Ltd. Gallic acid, disodium ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl) aminomethane (Tris), and phenol, all of analytical grade, were obtained from Tianjin Komiyo Reagent Co., Ltd. Standard substances of citric acid, tartaric acid, malic acid, lactic acid, succinic acid, and acetic acid, with a purity of ≥98%, were purchased from Sigma company, USA.

Instrumentation

The Agilent1260 high-performance liquid chromatography system was obtained from Agilent Technologies, USA. The ME-204E electronic analytical balance was sourced from Mettler-Toledo Instruments, Shanghai, China. The UV-752N ultraviolet–visible spectrophotometer was purchased from Shanghai Precision Scientific Instruments Co., Ltd., China. The CR-400 colorimeter was acquired from Konica Minolta, Japan. The PHS-3C pH meter was supplied by Shanghai Yidian Scientific Instrument Co., Ltd., China. The Biochrom30+ fully automated amino acid analyser was purchased from Biochrom Ltd., United Kingdom.

Preparation of PsR.–CpB. Enzyme

A total of 20 μl each of L. plantarum AS1.2437 and L. casei CICC21019 was spread onto MRS agar plates and incubated at 37 °C for 48 hr. A single colony exhibiting distinct characteristics was selected and inoculated into MRS broth for further activation, resulting in different lactic acid bacterial activation cultures with a concentration greater than 1.02 × 10⁸ CFU/ml. The activated two cultures were inoculated into MRS broth at a 3% (vol/vol) inoculum size and incubated at 37 °C with shaking at 150 r/min until reaching the late logarithmic growth phase (20 hr). Subsequently, inoculum cultures were prepared using different volume ratios of the two strains, with the viable cell count of lactic acid bacteria serving as the evaluation parameter. Through a single-factor experiment, a volume ratio of 2:1 was determined to be optimal for preparing the fermentation inoculum, which was then reserved as the seed culture for inoculation.

The dried rhizomes of Polygonatum sibiricum Red. were crushed to a particle size of less than or equal to 1.25 mm and mixed with dried C. pinnatifida Bunge according to a specific mass ratio. The mixture was soaked in a solvent-to-material ratio of 1:15 (mol/vol) at 60 °C for 30 min. Subsequently, 6.7% (mol/vol) brown sugar was added, and the mixture was blended for 5 min. The resulting mixture was placed in sterilised glass jars, and 4.5% (wt/wt) of a composite enzyme preparation (with a ratio of cellulase to pectinase of 2:1, mol/mol) was added. Enzymatic hydrolysis was conducted at 50 °C for 60 min, followed by sterilisation at 100 °C for 15 min. After cooling to room temperature, the fermentation substrate was obtained. According to the traditional medical formula of Polygonatum sibiricum Red. and C. pinnatifida Bunge herbal decoction with Artemisia scoparza Waldst.et Kit. (Ding et al., 1993), the mass ratio of Polygonatum sibiricum Red. to C. pinnatifida Bunge was maintained at 5:1, and a fermentation inoculum of 4% (vol/vol) was added. Fermentation was carried out in the dark at 34 °C for 52 hr. After fermentation, the mixture was filtered to obtain the PsR.–CpB. enzyme. To prevent contamination from unwanted microorganisms, all procedures were performed in a sterile environment.

Determination of microbial indices

The viable counts of lactic acid bacteria in the samples were determined according to GB 4789.35-2023 (National Health Commission of China & State Administration for Market Regulation of China, 2023). The detection of coliform bacteria was performed in accordance with GB 4789.3-2016 (National Health Commission of China & State Administration for Market Regulation of China, 2016a). The presence of Salmonella was assessed based on the guidelines outlined in GB 4789.4-2024 (National Health Commission of China & State Administration for Market Regulation of China, 2024). The determination of Staphylococcus aureus followed the protocol specified in GB 4789.10-2016 (National Health Commission of China & State Administration for Market Regulation of China, 2016b). Additionally, the count of moulds was conducted in accordance with GB 4789.15-2016 (National Health Commission of China & State Administration for Market Regulation of China, 2016a).

Determination of physicochemical indices

Determination of reducing sugars

The content of reducing sugars was determined using the 3,5-dinitrosalicylic acid (DNS) colorimetric method. To prepare the standard curve, aliquots of 0.20, 0.30, 0.40, 0.50, and 0.60 ml of an anhydrous glucose standard solution (1 mg/ml) were added to separate 10 ml test tubes. Each was diluted to 1 ml with distilled water. Then, 1.5 ml of DNS reagent was added to each tube, and the mixtures were thoroughly mixed. The tubes were heated in a boiling water bath for 5 min to develop colour, then allowed to cool to room temperature for 30 min. After cooling, the solutions were diluted to a final volume of 10 ml. The absorbance was measured at 550 nm, using a blank without glucose as a control. A standard curve was plotted with glucose mass on the X-axis and absorbance on the Y-axis, yielding a regression equation of Y = 1.3223X − 0.0588 and a correlation coefficient of R² = 0.9997. For the fermentation samples, the broth from different fermentation times was diluted as necessary. These samples were treated using the same procedure as the standards, and their absorbance was measured. The reducing sugar content in the fermentation samples, expressed as glucose equivalents, was calculated using the standard curve equation.

Determination of soluble solids content

The soluble solids content was measured at 25 °C using a handheld refractometer, with results expressed in degrees Brix (^oBrix). To begin, the light plate was opened, and the refractive prism was gently cleaned with a soft cloth. A few drops of distilled water were placed on the prism, and the light plate was carefully closed to ensure the liquid spread evenly across the prism surface. The instrument was then aligned with a light source or bright area, and the boundary line between light and dark was adjusted to the zero mark. After wiping away the distilled water, a glass rod was used to transfer drops of the fermentation liquid from different time points onto the test area. The refractometer was aligned to the light source, and the scale reading in the field of view was recorded for each sample.

Determination of total phenol content

The total phenol content was measured using the folin–phenol method. A gallic acid standard solution was prepared at a concentration of 1.0 mg/ml and then diluted to 0.1 mg/ml. From this diluted solution, aliquots of 0.3, 0.4, 0.5, 0.6, and 0.8 ml were transferred to 15 ml test tubes, and the volume was adjusted to 1.0 ml with deionised water. To each tube, 5 ml of 10% folin–ciocalteu reagent was added and mixed thoroughly. The mixture was allowed to stand for 5 min, after which 4 ml of 7.5% sodium carbonate solution (7.5 g/100 ml) was added. The reaction was carried out in the dark for 60 min. A blank control without gallic acid was prepared in the same manner. Absorbance was measured at 765 nm. A standard curve was plotted with the mass of gallic acid on the X-axis and absorbance on the Y-axis, resulting in a regression equation of Y = 9.1162X + 0.0298 and a correlation coefficient of R² = 0.9999. For the analysis of fermentation samples, the fermentation broth was diluted appropriately and treated using the same procedure. After processing, the absorbance was measured, and the total phenol content was calculated based on the standard curve. Each sample was measured in triplicate to ensure accuracy.

Determination of total flavonoid content

The total flavonoid content was measured using the NaNO₂-Al(NO₃)₃ colorimetric method, with rutin as the reference standard. A rutin standard solution was prepared at a concentration of 0.8 mg/ml. Aliquots of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ml of this solution were transferred into separate 25 ml volumetric flasks. Each flask received 1 ml of a 5% sodium nitrite (mol/vol) solution, followed by a 6-min rest period. Then, 1 ml of a 10% aluminium nitrate (mol/vol) solution was added, and the mixture was allowed to stand for another 6 min. Subsequently, 10 ml of a 4% sodium hydroxide (mol/vol) solution was added, and the solution was left for 20 min. Finally, the volume was adjusted to 25 ml with a 60% ethanol–water solution. A blank control without rutin was similarly prepared. The absorbance was measured at 510 nm. A standard curve was plotted with rutin mass on the X-axis and absorbance on the Y-axis, resulting in a regression equation of Y = 0.4574X − 0.0134 and a correlation coefficient of R² = 0.9999. For the analysis of fermentation samples, the broth from different fermentation times was processed using the same method. After treatment, the absorbance was measured, and the total flavonoid content was calculated using the standard curve. Each sample was measured in triplicate to ensure accuracy.

Determination of polysaccharide content

The polysaccharide content in the composite enzyme samples was determined using the phenol–sulphuric acid method. Soluble sugars, after dehydration with concentrated sulphuric acid, form furfural, which reacts with phenol to produce a coloured compound with a strong absorption peak at 490 nm. For the standard curve, aliquots of 0.2, 0.4, 0.6, 0.8, and 1.0 ml of a 0.2 mg/ml glucose standard solution were placed into 10 ml stoppered test tubes, and the volume was adjusted to 2 ml with distilled water. After mixing thoroughly, the tubes were placed in an ice-water bath. Then, 1.5 ml of a 4.5% phenol solution was added, mixed quickly, and followed by the addition of 6.5 ml of concentrated sulphuric acid. After mixing again and allowing the solution to cool, the tubes were incubated in a 40 °C water bath for 30 min. Subsequently, the tubes were placed in an ice-water bath for 5 min before returning to room temperature. A blank solution without glucose was prepared in the same manner. Absorbance was measured at 490 nm, and a standard curve was plotted with glucose mass on the X-axis and absorbance on the Y-axis, yielding a regression equation of Y = 4.489X + 0.0143 with a correlation coefficient of R² = 0.9992. For sample preparation, 6.25 ml of the fermentation broth was mixed with anhydrous ethanol to achieve an 80% alcohol content and left to stand overnight. The mixture was then filtered, and the supernatant was discarded. The residue was washed three times with 80% ethanol, dried at low temperature, dissolved in deionised water, and adjusted to a final volume of 250 ml to obtain the sample solution. This solution was treated using the same method as the standards, and the absorbance was measured to calculate the polysaccharide content in the enzyme samples.

Determination of total saponin content

The total saponin content was determined using the vanillin–perchloric acid colorimetric method. For samples taken at different fermentation times, 50 ml of each sample was mixed with an equal volume of water-treated saturated n-butanol. This mixture was extracted three times. The n-butanol phase was collected and evaporated to dryness using a rotary evaporator. The residue was then dissolved in anhydrous methanol and transferred to a 100-ml volumetric flask, where the volume was adjusted to obtain the total saponin solution for each sample, ready for analysis. The ginsenoside was used as the standard material, regression equation was Y = 1.5680X − 0.0686, and the correlation coefficient was 0.9963.

Determination of free amino acids

The determination of free amino acids in the samples was carried out using an automated amino acid analyser. The underlying principle involves the separation of amino acids in a column, followed by reaction with ninhydrin, with the resulting derivatives being detected via spectrophotometry to ascertain the concentration of each amino acid. For the procedure, 5.0 ml samples from various fermentation times were mixed with 5.0 ml of a 5% sulfosalicylic acid solution. This mixture was centrifuged at 6,000 rpm for 10 min. The supernatant was then evaporated to dryness using a rotary evaporator and redissolved in 1.0 ml of sodium citrate buffer. The solution was filtered through a 0.45-μm membrane before analysis. The chromatography was performed using a Na-type cation exchange resin column (4.6 mm × 200 mm, 3 μm particle size). The buffer solutions used included citrate buffer B₁ (pH = 3.20), B₂ (pH = 4.25), and B₅ (pH = 6.45). The buffer flow rate was set at 25.0 ml/h, with an injection volume of 20 μl. The reaction liquid flow rate was maintained at 10.0 ml/h, with the reaction chamber temperature at 138 °C and the column temperature at 65 °C. Quantification was achieved using an external standard method. Proline was detected at a wavelength of 440 nm, while other amino acids were detected at 570 nm.

Determination of pH value and total acid content

The pH value was measured with a pH meter. The total acid content was determined in strict accordance with the Chinese National Standard GB 12456-2021.

Determination of antioxidant activity

The activity of superoxide dismutase (SOD) was measured according to the method specified in the Chinese National Standard GB/T 5009.171-2003 (National Health Commission of China & Standardization Administration of China, 2003). Ferric ion-reducing antioxidant power (FRAP) assay: Samples from different fermentation times were diluted to a specific multiple for analysis. A volume of 0.2 ml of the diluted sample was mixed with 4.0 ml of the TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine) working solution and allowed to react for 10 min at 37 °C. The absorbance was then measured at 593 nm. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay: samples from different fermentation times were diluted to a specific multiple. A volume of 0.2 ml of the diluted sample was mixed with 3.8 ml of methanol solution of DPPH. After shielding from light for 30 min, the absorbance was measured at 517 nm. The 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) assay: samples from different fermentation times were diluted to a specific multiple. A volume of 0.2 ml of the diluted sample was mixed with 5.0 ml of ABTS working solution. After shielding from light for 6 min, the absorbance was measured at 734 nm.

Sensory evaluation

For the sensory evaluation of edible plant enzymes, a nine-point scoring system was employed based on relevant literature (Predieri et al., 2021). To ensure the reliability and accuracy of the sensory data, several precautionary measures and declarations were implemented and documented.

Participant selection and training

Ten students (five males and five females) with professional backgrounds in fermentation and sensory evaluation were carefully selected. A formal declaration was made to ensure that participants had no allergies or sensitivities to the substances being evaluated. They underwent a comprehensive training programme designed to standardise their understanding of the evaluation process and familiarise them with the specific attributes to be assessed.

Experimental environment control

The sensory evaluations were conducted in a controlled environment specifically designed to minimise external influences, as declared in the study protocol. The room was well-lit, odour-free, and maintained at a consistent temperature to ensure that participants’ assessments were based solely on the samples provided. Measures were taken to ensure silence and focus during evaluations, preventing any audio disturbances.

Sample preparation and presentation

Each sample of the PsR.–CpB. enzyme was prepared using standardised procedures to maintain consistency across evaluations. Samples were presented in identical, coded containers to prevent bias, and participants were instructed to cleanse their palates with water between assessments. A declaration was included to confirm that all samples were prepared fresh and handled under hygienic conditions to preserve their integrity.

Ethical considerations

The study was conducted following ethical guidelines, and approval was obtained from the relevant ethics committee (Approval No.: NYISTIRB2022-031). Participants provided informed consent, acknowledging their understanding of the evaluation process and their right to withdraw at any time without consequence. An explicit declaration was made regarding the confidentiality and anonymity of participant data.

Descriptive analysis

Descriptive analysis was utilised to assess the PsR.–CpB. enzyme, focusing on four evaluation attributes: colour, flavour, aroma, and organoleptic characteristics. The average scores were calculated, and the specific scoring criteria are detailed in Table 1.

Establishment of kinetic models

The kinetic properties of the fermentation of PsR.–CpB. enzyme from H. multiflorum were simulated using Origin 2019 software, based on the models presented in Table 2.

Data processing and analysis

Graphs and non-linear curve fitting were generated using Origin 2019 software. One-way analysis of variance was performed on the data using IBM SPSS Statistics 26.

Results and discussion

Microbiological analysis

To assess the microbiological safety of the PsR.–CpB. enzyme product, probiotic lactic acid bacteria and foodborne pathogenic bacteria were evaluated in the final product. The results were assessed according to the current microbial index standards for edible plant enzymes in China. The viable count of probiotic lactic acid bacteria in the PsR.–CpB. enzyme reached 3.42 × 10⁹ CFU/ml at the end of fermentation, significantly exceeding the standard requirement of 10⁵ CFU/ml, demonstrating excellent probiotic characteristics. No food-borne pathogenic bacteria were detected before or after fermentation, indicating effective sterilisation of the fermentation substrate prior to processing (Supplementary Table 1). This also suggests that the fermented product possesses favourable edible and safety qualities, complying with the microbial safety limits set forth in the standards for edible enzymes, which positively impacts its market development.

Physicochemical analysis of PsR.–CpB. enzyme

The results of the physicochemical properties of the PsR.–CpB. enzyme before and after fermentation are presented in Table 3. Lactic acid bacteria, which play a key role in enzyme preparation, convert carbohydrates and other substances in the substrate into organic acids such as lactic acid and citric acid through their metabolic activities. Consequently, the soluble solid content, pH value, and reducing sugar content of the enzyme significantly decreased (p < .05) at the end of fermentation, while the total acid content significantly increased (p < .05). Additionally, the resulting suitable sugar-to-acid ratio imparts a mild and refreshing taste to the enzyme product. Studies have shown that the dynamic changes in the sugar-to-acid ratio during fermentation are related to factors such as substrate composition, microbial species, inoculation level, and fermentation duration (Garcia et al., 2018). Compared to the pre-fermentation samples, the enzyme product at the end of fermentation exhibited significant increases in functional activity substances, including SOD activity, total flavonoid content, total phenolic content, total saponin content, and polysaccharide content (p < .05). This indicates that the fermentation activity of lactic acid bacteria promotes the production, conversion, and release of active components such as SOD, phenolic compounds, and polysaccharides from the substrate, with SOD activity increasing by approximately twofold. The acidic environment generated during fermentation positively influences the flavour of the enzyme and exerts antimicrobial effects. Additionally, it plays a protective role in the metabolic accumulation of phenolic compounds and other bioactive substances, such as SOD. Most lactic acid bacteria that proliferate during fermentation are probiotics, and their co-consumption alongside active compounds can promote human health (Saud et al., 2024). Polysaccharides are important active components of Polygonatum sibiricum Red., and studies have found that they are the primary contributors to the sweetness of Polygonatum sibiricum Red. products, facilitating greater consumer acceptance of foods predominantly made from Polygonatum sibiricum Red. (Wang et al., 2017). With the conclusion of fermentation, the quality of the enzyme product tends to stabilise, and all physicochemical indices meet the specifications outlined in relevant standards.

Free amino acid analysis

Composition and content analysis

Free amino acids are important components of edible plant enzymes and possess significant nutritional value for human health. As shown in Table 4, a total of 17 amino acids were detected in the PsR.–CpB. enzyme before and after fermentation, including 7 essential amino acids (EAAs) and 10 non-essential amino acids (NEAAs). Following fermentation by lactic acid bacteria, the total amino acids (TAAs) content and the total non-essential amino acid content in the PsR.–CpB. enzyme decreased by 0.57% and 4.96%, respectively. Among the EAAs, the contents of Val, Leu, Ile, and Met were all reduced compared to pre-fermentation levels. This reduction may be attributed to two main factors. Firstly, the vigorous growth and metabolic activity of lactic acid bacteria during fermentation may result in their utilisation of amino acids exceeding the proteolytic activity produced by proteases (Wang et al., 2021). Secondly, during fermentation, amino acids may undergo partial oxidation, enzymatic conversion, or participate in the synthesis of other volatile flavour compounds (Guo et al., 2024). For instance, EAAs such as Ile, Leu, and Val can contribute to the formation of volatile aroma compounds in the enzyme, while certain non-essential amino acids can be enzymatically converted into other substances through specific enzymes produced by lactic acid bacteria. Ser can be catalytically transformed to pyruvate by serine deaminase generated during lactic acid fermentation, leading to a decrease in its content. Similarly, arginine, which had the highest content pre-fermentation, can be enzymatically converted to carbon dioxide, ammonia, and ornithine by arginine deiminase produced by lactic acid bacteria.

However, post-fermentation, the total amount of EAAs in the PsR.–CpB. enzyme increased by 4.86%, with Thr, Phe, and Lys showing significant increases compared to the pre-fermentation samples (p < .05). This increase may be attributed to the death of bacterial cells in the later stages of fermentation, which leads to the degradation of cellular protein. This finding indicates that lactic acid fermentation enhances the nutritional value of amino acids in the PsR.–CpB. enzyme. In summary, lactic acid fermentation results in an increase in the EAAs/TAAs and EAAs/NEAAs ratios, thereby improving the nutritional value of the PsR.–CpB. enzyme, which is significant for enhancing product quality.

Analysis of therapeutic amino acids

Amino acids such as Asp, Glu, Gly, Met, Leu, Phe, Tyr, Lys, and Arg are found in relatively low concentrations in plants from nature, and some of these cannot be synthesised by the human body. They are necessary for the nitrogen balance in the body and are collectively referred to as therapeutic amino acids. These amino acids play essential physiological roles and possess certain medicinal values. For instance, Glu is involved in the treatment of nerve damage, concussions, and epilepsy; it readily combines with blood ammonia to form glutamine, which can alleviate ammonia toxicity and protect the liver, as well as treat and prevent hepatic encephalopathy (Luttenbacher et al., 2022). Lys promotes the function of the central nervous system and supports growth and development (Hosseinzadeh et al., 2020). Asp is effective in treating chronic liver diseases, enhancing liver function, protecting the liver, preventing heart disease, and aiding in recovery from fatigue.

Figure 1 illustrates the changes in the composition and content of therapeutic amino acids in the samples before and after fermentation. As indicated in Figure 1, the contents of Glu, Tyr, Phe, and Lys among the nine therapeutic amino acids significantly increased after fermentation by lactic acid bacteria compared to pre-fermentation levels (p < .05), whereas the remaining five therapeutic amino acids experienced a decrease. Nonetheless, the total proportion of therapeutic amino acids to TAAs significantly increased after fermentation (p < .05), rising from 50.88% before fermentation to 53.59% after fermentation. Among these, Leu, Lys, and Phe are essential amino acids that are indispensable for the body. Arg, which had the highest concentration among therapeutic amino acids both before and after fermentation, is an essential amino acid for infants (Cao et al., 2016). Its major medicinal value and physiological functions include the protection of cardiovascular and cerebrovascular health, improvement of related diseases, enhancement of liver function, and boosting immunity (Hu et al., 2020). In summary, the lactic acid fermentation process led to changes in the content and proportion of different therapeutic amino acids in the hawthorn enzyme; however, the proportion of therapeutic amino acids relative to TAAs increased, indicating a favourable medicinal value that positively impacts the quality of the final product.

Organic acid analysis

Organic acids are one of the key indicators for evaluating enzyme quality, significantly affecting storage stability, sensory acceptability, and nutritional characteristics. They not only impart unique flavours to the enzymes but also serve as carbon sources and electron donors during microbial fermentation. Additionally, organic acids exhibit antibacterial and anti-inflammatory properties, promote digestion, and help maintain acid–base balance. Figure 2 illustrates the changes in organic acid content and composition in samples before and after fermentation. As shown, the primary organic acids in the pre-fermentation samples were citric acid and malic acid, with lactic acid not detected. Following fermentation, the predominant organic acids in the resulting enzyme were lactic acid, acetic acid, and tartaric acid, while malic acid was not detected. The content of all six organic acids underwent significant changes (p < .05) after fermentation, indicating that lactic acid fermentation greatly altered the content and types of organic acids in the substrate.

Acid production during fermentation is a critical characteristic of enzyme production. Different fermentation methods, raw materials, and microbial strains result in varying contents and types of organic acids in the enzyme products, thereby contributing to diverse flavours. Among the main organic acids before fermentation, malic acid is noted for its strong and pungent flavour, which becomes irritating at higher concentrations (Peng et al., 2021), while citric acid is an important organic acid in fruits and vegetables, characterised by a strong sour taste (Zhu et al., 2016). Both acids significantly contribute to the flavour of the substrate prior to fermentation. Their concentrations decreased significantly (p < .05) upon completion of fermentation, with malic acid nearly disappearing. In contrast, lactic acid increased from undetectable levels before fermentation to 4.43 g/L after fermentation, becoming the most abundant organic acid in the final product. Lactic acid is associated with a milder sour taste, positively influencing the final product. During the fermentation process, lactic acid bacteria may convert malic acid and citric acid into lactic acid through metabolic pathways such as the malic acid–lactic acid pathway and the tricarboxylic acid cycle (Călugăr et al., 2024). This conversion leads to a reduction in the concentrations of malic and citric acids by the end of fermentation, accompanied by an increase in lactic acid content. In addition to lactic acid, other major organic acids in the final enzyme product, such as acetic acid and tartaric acid, also contribute to the flavour profile of the enzyme (Li et al., 2023b). Moderate levels of acetic acid can impart a pleasant vinegar aroma, while tartaric acid provides a sweet flavour. The combination of these acids plays a significant role in influencing the overall flavour of the final product.

Sensory quality analysis

The sensory qualities of the PsR.–CpB. enzyme before and after inoculation with lactic acid bacteria were analysed, and the results are shown in Figure 3. As illustrated in Figure 3, the colour, flavour, aroma, and organoleptic characteristics of the enzyme improved after lactic acid bacteria fermentation compared to the pre-fermentation samples. Notably, the improvement in organoleptic characteristics was particularly significant, with scores rising from 2.00 to 8.67. The flavour enhancement was also pronounced; the pre-fermentation samples exhibited the distinctive off-flavours typical of medicinal herbs, which disappeared after fermentation, greatly enhancing palatability. The final product was overall clear and bright, with no suspensions or sediments. The pre-fermentation samples exhibited a darker colour, which transformed into an overall red-brown hue after fermentation. Aroma played a crucial role in the overall perception of a beverage, significantly influencing consumer preference and acceptance (Bora et al., 2023). Following the fermentation process, the aroma profile of the beverage underwent a notable transformation, becoming more harmonious and mellow. This enhancement in aromatic quality contributed to a substantial improvement in the overall sensory evaluation. Quantitatively, the sensory score experienced a remarkable increase, rising from 12.35 prior to fermentation to 34.20 post-fermentation.

Changes of total phenolic and flavone contents

Total phenols and flavonoids are closely linked to the antioxidant activity of enzymes, and phenolic compounds also offer anti-inflammatory, anti-ageing, neuroprotective, and immunomodulatory benefits. As shown in Figure 4, during fermentation, the total phenol content initially increases, then decreases, and rises again, while the total flavonoid content shows an initial increase followed by a decrease. By the end of fermentation, the total phenol and flavonoid contents reach 941.30 and 864.92 mg/L, respectively, which are significantly higher than those before fermentation (p < .05). This indicates that lactic acid bacteria fermentation can markedly enhance the total phenol and flavonoid content in PsR.–CpB. enzyme. Furthermore, similar results have been reported in other studies. For instance, Kwaw et al. used Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus paracasei to ferment mulberry juice, resulting in significant increases in total phenol and flavonoid content compared to pre-fermentation levels. They also noted significant differences in fermentation outcomes between different lactic acid bacterial strains, likely due to variations in the strains’ adaptability to the substrate and their enzyme production capabilities (Kwaw et al., 2018). Similarly, Yan et al. observed that co-fermentation of blueberry pomace with Lactobacillus rhamnosus and L. plantarum significantly increased the total phenol and flavonoid content, enhancing the antioxidant and anti-fatigue properties of the fermented blueberry pomace (Yan et al., 2019). The increase in total phenols and flavonoids during fermentation may result from enzymes produced by lactic acid bacteria breaking down plant tissues in the substrate, such as cell walls. The high sugar concentration in the initial substrate and the osmotic environment created by acidic metabolic products may further release phenolic compounds or convert bound and macromolecular phenols into free, low-molecular-weight phenols, increasing their levels. Conversely, the decrease in phenolic content might be due to adsorption, precipitation, or oxidation during fermentation, as phenolic compounds can inhibit microbial growth when reaching certain concentrations, prompting microbes to produce substances that degrade phenols (Khan et al., 2018). In summary, the changes in phenolic compounds during fermentation are influenced by raw materials and microbial interactions.

Kinetic analysis

To further investigate the variations in fermentation characteristics and patterns during the lactic acid bacteria fermentation of PsR.–CpB. enzyme, three classic S-shaped models including Bohzman, Logistic, and SGompertz were employed for non-linear fitting of the data pertaining to reducing sugars, total acids, and viable lactic acid bacteria counts throughout the fermentation process. By comparing the correlation coefficients, the model that best fit the fermentation data was selected to describe the fermentation dynamics. Kinetic models for the lactic acid bacteria fermentation of PsR. –CpB. Enzyme were established, thereby providing a theoretical foundation for enhancing PsR.–CpB. enzyme product quality, optimising fermentation process parameters, and achieving industrial-scale production.

Kinetic model of reducing sugar metabolism

The data on reducing sugar content changes during the fermentation was counted, along with the fitting results of three classic models was calculated (Supplementary Table 2). The changes in reducing sugar content during fermentation were measured, and the fitting results of three classical models were calculated (Supplementary Table 2). The Boltzmann model demonstrated the highest correlation coefficient (R²) of 0.9975, surpassing the other two models and exhibiting superior fitting accuracy. Subsequently, the Boltzmann model was employed to perform non-linear fitting of the reducing sugar content variation curve throughout the fermentation process (Supplementary Figure 1). The Boltzmann model’s fitted curve exhibited an extremely high concordance with the reducing sugar content change curve, indicating the model’s robust predictive capability for reducing sugar dynamics within the substrate during fermentation. The Boltzmann kinetic equation is as follows:

In contrast, the SGompertz model’s correlation coefficient was −1.4897, suggesting its unsuitability for characterising the variation pattern of reducing sugars during the fermentation.

Kinetic model of total acid content

The changes in total acid content during fermentation were measured, and the fitting results of three classical models were calculated (Supplementary Table 3). The Boltzmann model exhibited the highest correlation coefficient (R²) of 0.9984, significantly outperforming the other two models and demonstrating the most optimal fitting precision. A subsequent non-linear fitting of the total acid content variation curve using the Boltzmann model was conducted (Supplementary Figure 2). The fitted curve demonstrated an exceptionally high concordance with the actual total acid content change curve, substantiating the Boltzmann model’s robust predictive capacity for tracking total acid content dynamics within the substrate throughout the fermentation process. The Boltzmann kinetic equation is as follows:

Kinetic model of viable lactic acid bacteria count

The variations of viable lactic acid bacteria count during fermentation were quantified, and the fitting results of three classical models were determined (Supplementary Table 4). The Boltzmann model achieved a correlation coefficient (R²) of 0.9975, surpassing the other two models and demonstrating good fitting accuracy. Subsequently, the Boltzmann model was applied to perform non-linear fitting of the variation curve of viable lactic acid bacteria counts throughout the fermentation process (Supplementary Figure 3). The fitted Boltzmann curve exhibited an exceptionally high concordance with the actual changes in viable lactic acid bacteria counts, indicating that the Boltzmann model effectively predicts the dynamics of lactic acid bacteria counts within the fermentation system. This model adequately reflects the growth patterns of lactic acid bacteria during the fermentation of PsR.–CpB. enzyme. Furthermore, it provides a foundational basis for further exploring the relationships between lactic acid bacteria, metabolic products, and their functional roles during the fermentation process. The Boltzmann kinetic equation is as follows:

Antioxidant activity

Changes of antioxidant activity

The antioxidant activity of PsR.–CpB. enzyme products were evaluated using three commonly employed indicators: FRAP, DPPH, and ABTS radical scavenging capacities. These assays reflect the changes in antioxidant capability during the fermentation process by lactic acid bacteria. As illustrated in Figure 5, all three antioxidant indicators showed significant enhancement after lactic acid bacterial fermentation (p < .05). The FRAP value exhibited an overall fluctuating upward trend, increasing from 163.88 to 206.91 mmol Trolox/L by the end of fermentation. In contrast, the DPPH and ABTS radical scavenging capacities displayed a pattern of increase–decrease–increase during fermentation. At the conclusion of the fermentation process, the DPPH value increased from 46.20 to 92.14 mmol Trolox/L, while the ABTS value rose from 60.24 to 95.45 mmol Trolox/L. Notably, the three indicators representing antioxidant capacity showed significant increases primarily at 24- or 32-hr post-fermentation, followed by subsequent fluctuations in values. At the end of the fermentation process, the antioxidant activity indicators increased by 99.44% (DPPH), 58.45% (ABTS), and 26.26% (FRAP), respectively, compared to the pre-fermentation levels. These results indicate that while lactic acid fermentation enhanced all three antioxidant indicators, the effects varied among the different metrics. Specifically, the DPPH radical scavenging capacity exhibited the most substantial increase, followed by the ABTS cation radical scavenging capacity, with the least enhancement observed in the iron ion reducing capability.

Correlation analysis of antioxidant activity and bioactive substances

Lactic acid bacterial fermentation of plant enzymes typically exhibits enhanced antioxidant activity, which can be attributed to two main factors. Firstly, lactic acid bacteria possess inherent antioxidant capabilities; when they exist in environments rich in free radicals, they can produce active substances such as SOD and catalase, which effectively scavenge free radicals. Secondly, during the fermentation of fruit and vegetable substrates, lactic acid bacteria can release antioxidant active components from the raw materials and generate new active substances, such as polyphenols, flavonoids, and polysaccharides, contributing to their enhanced antioxidant activity.

As illustrated in Table 5, the fermentation process of PsR.–CpB. enzyme revealed significant correlations between three key active substances (total phenols, total flavonoids, and polysaccharides) and antioxidant capacities. Notably, total phenols demonstrated extremely significant correlations (p < .01) with four antioxidant indicators: SOD activity, DPPH, ABTS+, and FRAP. Total flavonoids similarly exhibited extremely significant correlations (p < .01) with SOD activity, DPPH, and FRAP. Polysaccharide content showed significant correlations (p < .05) with three antioxidant indicators. These findings indicate that increased total phenol and flavonoid contents directly enhance antioxidant capabilities through two primary mechanisms: post-fermentation elevation of phenolic and flavonoid concentrations, and potential generation of highly potent phenolic and flavonoid compounds during fermentation. Wang et al. (2022) previously reported that lactobacilli fermentation of kiwifruit juice significantly improved FRAP, DPPH, and ABTS⁺ radical scavenging abilities (p < .05), concurrent with the formation of two novel phenolic compounds—a observation consistent with our preliminary hypotheses. Consequently, the variations in antioxidant activity of PsR.–CpB. enzyme are not solely determined by total phenolic and flavonoid quantities but also significantly influenced by the structure and composition of newly generated phenolic and flavonoid molecules during fermentation. Although polysaccharide content demonstrated significant correlations with antioxidant indicators (p < .05), its impact remained relatively modest, potentially attributed to the specific structural and functional characteristics of fermentation-generated polysaccharides. In conclusion, the active substances produced through lactobacilli fermentation enhance antioxidant performance not only quantitatively but also through critical structural and functional modifications. This comprehensive investigation provides substantial theoretical foundations for further exploring the health benefits of fermented products and developing innovative functional foods with enhanced antioxidant properties.

Conclusion

This study successfully developed a kinetic model for the fermentation of Polygonatum sibiricum Red. and Crataegus pinnatifida Bunge utilising lactic acid bacteria. A systematic analysis was conducted on the physicochemical properties and antioxidant capabilities of the resulting fermented product. Dynamic monitoring during the fermentation process revealed that changes in reducing sugars, total acidity, and viable lactic acid bacteria counts could be effectively modelled using the Boltzmann equation, achieving fitting coefficients (R²) greater than 0.99, which indicates the high reliability of the model. Upon completion of fermentation, significant increases (p < .05) were observed in total phenols, total flavonoids, SOD activity, and polysaccharide content. Additionally, the enhancement of essential and medicinal amino acids suggests an improved nutritional profile. Following lactic acid fermentation, substantial improvements were noted in the antioxidant activities assessed through the FRAP, DPPH, and ABTS assays. Correlation analysis demonstrated a significant positive relationship between the concentrations of total phenols, polysaccharides, and antioxidant capacity. The results of this research underscore the medicinal potential and antioxidant properties derived from the fermentation of Polygonatum sibiricum Red. and C. pinnatifida Bunge, laying a vital theoretical foundation and practical framework for the development of functional foods informed by traditional medicine and dietary practices. Future research will focus on optimising fermentation strategies with different strains of lactic acid bacteria and varying fermentation conditions to further enhance the quality and bioactivity of the produced enzymes.

Data availability

The data that support the findings of this study are available on request from the corresponding author.

Author contributions

Yanli Ma (Conceptualization [equal], Funding acquisition [equal], Writing—review & editing [equal]), Yin-zhuang Wang (Data curation [equal], Software [equal]), Yuan Feng (Investigation [equal], Methodology [equal]), Ding-ding Duan (Validation [equal]), Haiyan Yang (Formal analysis [equal], Methodology [equal]), Jun Wang (Conceptualization [equal], Funding acquisition [equal], Writing—original draft [equal])

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Funding

This work was supported by the Youth Backbone Training Program of Higher Education Institutions in Henan Province (2020GGJS226), the Key Research and Development Project of Tibet (XZ202101ZY0015G), and the Major Collaborative Innovation Project of Nanyang (21XTCX12005).

References