Abstract

This study aimed to develop a Lactobacillus rhamnosus-fermented Polygonati Rhizoma and Cistanches Herba compound oral liquid, with sensory score as the core evaluation index. Single-factor tests combined with response surface methodology were used to optimize fermentation parameters, exploring the effects of Polygonati Rhizoma to Cistanches Herba volume ratio, inoculum size, and fermentation time on product flavor. Meanwhile, in vitro antioxidant activity was evaluated via DPPH and ABTS⁺ free radical scavenging assays.

The optimal fermentation conditions were determined as follows:

Polygonati Rhizoma to Cistanches Herba volume ratio: 21:19

Inoculum size: 8.04 × 10⁶ CFU/mL

Fermentation time: 31 h

Under these conditions, the sensory score reached 89.1. The fermented oral liquid contained total acid 3.85 mL/100 mL, reducing sugar 31.84 mg/mL, polysaccharides 2.97 mg/mL, flavonoids 0.65 mg/mL, and polyphenols 0.80 mg/mL. Its DPPH and ABTS⁺ free radical scavenging rates were 75.0% and 62.7%, respectively, demonstrating strong antioxidant activity.

Key words: Polygonati Rhizoma; Cistanches Herba; Lactobacillus rhamnosus; fermentation process optimization; antioxidant activity; how to optimized cistanche benefits

 

Cistanche Dietary Supplement Anti-Oxidation Support Phenylethanoid Glycosides 75% Echinacoside 30% Acteoside 12%

 

 

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1. Introduction

1.1 Polygonati Rhizoma (Huangjing)

Polygonati Rhizoma is the dried rhizome of Polygonatum kingianum Coll. et Hemsl., Polygonatum sibiricum Red., or Polygonatum cyrtonema Hua (Liliaceae). It is sweet in taste and neutral in nature, with well-documented TCM benefits: replenishing Qi and nourishing Yin, invigorating the spleen, moistening the lungs, and tonifying the kidney. Rich in polysaccharides, flavonoids, saponins, and other bioactive components, it supports immune regulation, antioxidant defense, anti-aging, anti-fatigue, and anti-inflammatory effects. In clinical practice, it is often combined with Cistanches Herba to address kidney essence deficiency, lumbar and knee weakness, fatigue, and spleen-stomach weakness.

1.2 Cistanches Herba (Rou Cong Rong) - Premium Cistanche tubulosa Extract

Cistanches Herba is the dried fleshy stem with scale leaves of Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) Wight. Our factory specializes in high-purity Cistanche tubulosa extracts with markedly higher active ingredient content (e.g., echinacoside, verbascoside, polysaccharides) than conventional Cistanche deserticola products, making it far more potent for health applications.

Recorded in Shennong's Classic of Materia Medica, Cistanches Herba "treats five consumptions and seven damages, tonifies the interior, eliminates cold and heat pain in the lower abdomen, nourishes the five zang-organs, strengthens Yin, and replenishes essence". Modern research confirms its polyphenols, polysaccharides, and phenylethanoid glycosides deliver kidney-yang warming, essence-blood replenishing, immune enhancement, and robust antioxidant effects.

 

Cistanche Active Ingredient- Echinacoside

 

 

 

1.3 Probiotic Fermentation + Traditional Chinese Medicine

Modern fermentation technology has unlocked new potential for herbal medicines. Probiotic fermentation with lactic acid bacteria boosts the release of active components, reduces toxic constituents, enhances efficacy, balances gut microbiota, and strengthens immunity. Studies show Lactobacillus rhamnosus fermentation elevates levels of flavonoids, polysaccharides, and polyphenols in herbs, directly lifting antioxidant and bioactivity.

 

Table 1. Factors and levels of response surface experiment

Level	A: Ratio of Astragalus membranaceus to Poria cocos	B: Inoculum size (CFU/mL)	C: Fermentation time (h)
-1	12:28	6.24×1066.24 \times 10^{6}6.24×106	12
0	20:20	6.24×1076.24 \times 10^{7}6.24×107	24
1	28:12	6.24×1086.24 \times 10^{8}6.24×108	36

 

This study focuses on how to optimized cistanche benefits via Lactobacillus rhamnosus fermentation of a Polygonati Rhizoma–Cistanches Herba compound, using sensory quality and antioxidant activity as benchmarks. The goal is to establish a scalable, high-efficacy fermentation process for developing premium Cistanche-based dietary supplements tailored for Western consumers.

Table 2. Sensory scoring criteria for Polygonati Rhizoma and Cistanches Herba compound oral liquid

Item	Scoring criteria	Score range
Physical state	Uniform, no layering, no precipitation	11–15
	Relatively delicate, no layering, slight precipitation	6–<11
	Non-uniform, slight layering, heavy precipitation	0–<6
Color/luster	Uniform color, glossy, bright red	16–20
	Relatively uniform, luster not obvious, darker color	10–<16
	Non-uniform, no luster, color too light or too dark	0–<10
Aroma/odor	Uniform aroma, no off-odor, caramel-like aroma	16–25
	Light aroma, light caramel-like aroma, slightly sour	6–<16
	Unbalanced aroma, overly sour, off-odor	0–<6
Taste	Sweet–sour balanced, delicate mouthfeel	31–40
	Slightly sweet or slightly sour, relatively delicate mouthfeel	16–<31

 

Cistanche Active Ingredient- Echinacoside

2. Materials and Methods

2.1 Materials and Reagents

Polygonati Rhizoma: Anhui Shangfengyuan Health Technology Co., Ltd.

Cistanches Herba: Premium Cistanche tubulosa (our factory extract, higher active content)

Lactobacillus rhamnosus: Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University

Analytical-grade reagents: DPPH, ABTS, rutin, gallic acid, glucose, phenol, sulfuric acid, etc.

MRS broth, standard culture media

2.2 Instruments

Microplate reader (BioTek), UV-Vis spectrophotometer (UV-5200), laminar flow cabinet, autoclave (GF54DA), CO₂ incubator (MCO-170), electronic balance (FA2204)

2.3 Experimental Procedures

2.3.1 Process Flow

Herbal raw materials → Extraction → Mixed extract → Sterilization → Inoculation & fermentation → Filtration & sterilization → Packaging → Finished product

2.3.2 Preparation of Extracts

Polygonati Rhizoma and Cistanche tubulosa were soaked for 30 min, then extracted twice at gentle boiling (1 h each). The raw material concentration of both extracts was 6.25 g/100 mL.

2.3.3 Fermentation

Extracts were blended at set ratios, sterilized (121 °C, 20 min), inoculated with Lactobacillus rhamnosus, and fermented at 37 °C.

2.3.4 Single-Factor Tests

Variables tested:

Polygonati Rhizoma : Cistanches Herba volume ratios (12:28, 16:24, 20:20, 24:16, 28:12)

Inoculum levels (6.24×10⁵, 6.24×10⁶, 6.24×10⁷, 6.24×10⁸, 6.24×10⁹ CFU/mL)

Fermentation times (12, 24, 36, 48, 60 h)

 

Table 5. Content of components of Polygonati Rhizoma and Cistanches Herba compound oral liquid

Sample	Total acid (mL/100 mL)	Reducing sugar (mg/mL)	Polysaccharides (mg/mL)	Astragaloside IV (mg/mL)	Total flavonoids (mg/mL)
Unfermented	3.15 ± 0.11	21.06 ± 0.20	2.88 ± 0.02	0.55 ± 0.01	0.68 ± 0.02
Fermented	3.85 ± 0.03	31.84 ± 0.17	2.97 ± 0.07	0.65 ± 0.01	0.80 ± 0.04

2.3.5 Response Surface Methodology (RSM)

A 3-factor, 3-level Box–Behnken design was applied, with sensory score as the response. Independent variables:

A = Polygonati Rhizoma : Cistanches Herba ratio

B = Inoculum size

C = Fermentation time

2.3.6 Evaluation Methods

Sensory evaluation: 10 trained panelists scored texture, color, aroma, and taste (max 100 points).

Physicochemical indexes: Total acid (GB/T 12456-2021), reducing sugar, polysaccharides, flavonoids, polyphenols.

Antioxidant activity: DPPH and ABTS⁺ free radical scavenging rates.

Microbiological safety: E. coli, molds, and yeasts tested per GB 4789 series.

 

Cistanche Active Ingredient- Acteoside

3. Results and Discussion

3.1 Single-Factor Test Results

Ratio effect: Sensory scores first rose then fell. The 20:20 ratio yielded balanced sweetness/sourness, bright reddish color, and harmonious caramel aroma.

Inoculum effect: Scores peaked at 6.24×10⁷ CFU/mL, with ideal flavor and mouthfeel.

Time effect: 24 h fermentation gave the best sensory profile; longer times led to excessive acidity.

3.2 Response Surface Optimization

The regression model was highly significant (P < 0.0001) with excellent fit (R² = 0.9913). Factor impact order: inoculum size > fermentation time > herb ratio, consistent with how to optimized cistanche benefits via controlled fermentation.

Optimal conditions verified:

Ratio 21:19, inoculum 8.04×10⁶ CFU/mL, 31 h fermentation

Actual sensory score: 89.1 ± 3.3 (close to predicted value), confirming process reliability.

3.3 Physicochemical and Bioactive Enhancements

Fermentation significantly increased total acid, reducing sugars, polysaccharides, flavonoids, and polyphenols. Lactobacillus rhamnosus breaks down cell walls, hydrolyzes macromolecules, and boosts bioavailability - directly optimizing cistanche benefits for human absorption.

3.4 Antioxidant Activity Improvement

DPPH scavenging rate: 75.0%

ABTS⁺ scavenging rate: 62.7%

Fermentation markedly elevated antioxidant capacity compared to unfermented extract, driven by higher levels of reducing sugars, flavonoids, and polyphenols.

3.5 Microbiological Safety

No E. coli, molds, or yeasts detected. The product fully meets international food safety standards.

Fig.5 DPPH and ABTS+ free radical scavenging rates of fermented Polygonati Rhizoma and 13 Cistanches Herba compound oral liquid

 

4. Conclusion

This study defines a science-backed, repeatable method for how to optimized cistanche benefits through Lactobacillus rhamnosus fermentation. The optimal process yields a safe, great-tasting compound oral liquid with superior antioxidant activity and enhanced bioactive components.

Our factory's premium Cistanche tubulosa extract - with higher active ingredient content - ensures this fermented product delivers reliable, potent health benefits aligned with Western consumer expectations for immunity, vitality, kidney support, and anti-aging.

This technology provides a solid foundation for developing innovative, evidence-based Cistanche dietary supplements for global markets.

 

Factory Information

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We specialize in high-purity Cistanche tubulosa extracts with industry-leading active component levels, ideal for creating premium, science-backed health products.