Co-fermentation and post-processing: development of a novel myrtle rice beverage, and analysis of its product characteristics

ABSTRACT

To fully develop the medicinal and edible value of myrtle and improve the market visibility, myrtle berries and glutinous rice were used as raw materials and co-fermented using rice leaven. Uniform design method was used to determine the optimal fermentation conditions. The co-fermentation product was then underwent post-processing, including blending, grinding and homogenization, to produce a novel myrtle rice beverage, which was analyzed for product characteristics. Results showed that the optimal fermentation conditions were a myrtle berries of 24%, a rice leaven of 1%, a water addition amount of 100%, a fermentation temperature of 22°C, a fermentation time of 96 h. The co-fermented product was diluted 1:3 with water, ground, and homogenized with 0.1% agar and 0.2% xanthan gum to produce the final myrtle rice beverage. The beverage boasts a low sugar and alcohol content, along with a moderate level of acidity, making it a healthy beverage with antioxidant properties.

GRAPHICAL ABSTRACT

KEYWORDS:

• Co-fermentation
• post-processing
• myrtle rice beverage
• product characteristics

1. Introduction

Myrtle is a perennial evergreen and fragrant shrub that belongs to the Myrtaceae, the shrub is indigenous to the Mediterranean and the Middle East, and also broadly distributed in southern China (Mele et al., 2019; Usai et al., 2020). Myrtle leaves, fruits, flowers, branches, and roots contain a variety of bioactive compounds, which make it a valuable resource in industries such as perfumery, cosmetics, pharmaceuticals, and food production (Ghafouri & Rahimmalek, 2018; Tayeh et al., 2017). Especially myrtle berries contain more bioactive substances, including flavonoids, polyphenols, anthocyanin, and triterpenes, endowing it with therapeutic potential for ailments such as diabetes, hypertension, anti-inflammatory, antiviral, antioxidant, and neuroprotective properties, etc (Hennia et al., 2019; Medda et al., 2021; Siracusa et al., 2019). Myrtle berries has two diferent colors, black or white, the black fruit mainly contains polyphenols with high antioxidant activity, and the white fruit contain unsaturated fatty acids such as myrtenyl acetate, linoleic acid, and oleic acid (Giampieri et al., 2020; Gorjian & Khaligh, 2023; Siracusa et al., 2019). In addition to its medicinal properties, it is also rich in nutritional value, such as high protein and sugar content, low fat levels, an array of essential elements, amino acids, and vitamins, rendering it suitable for the production of healthy food (Lai et al., 2015).

The ripe myrtle berries is edible and has been used to aromatize wine, confectionery, and sweet dishes, to produce liquors and to replace black pepper as spice for meats and sauces (Babou et al., 2016; Gorjian & Khaligh, 2023). However, myrtle berries suffers from certain drawbacks, including a short maturation period, limited pulp content, numerous seeds, and perish ability, resulting in poor taste and difficulty in preservation. In addition, it is not well recognized in the marketplace and is far removed from other berry groups, such as blueberries and Aronia melanocarpa. A mere 10% of the fruit remains suitable for fresh consumption, while the remaining 90% is discarded, leading to waste and hampering its potential for value-added processing (Huang et al., 2010; Luo et al., 2015). Currently, myrtle berries is used primarily in flavor enhancers and liquors, its full potential remains underexplored, urgent need to explore new avenues of utilization, increasing utilization of resources and social acceptance.

Fermented glutinous rice (FGR), a rice-based alcoholic beverage produced through rice leaven fermentation, contains amino acids, peptides, lactic acid, and trace elements (Wen et al., 2019; Yang et al., 2022). It promotes digestive gland secretion, enhances appetite, boosts immunity, and improves blood circulation (Surojanametakul et al., 2019; Wang et al., 2018). FGR is easy to prepare and has a short production cycle, it’s very popular in Asia, especially in China and Japan, owing to its low ethanol content, sweet and refreshing taste, and nutritional benefits. Nevertheless, it lacks diversity in product type, is primarily used as a food accessory ingredient, has limited application scenarios, and is less prevalent as a standalone beverage. Furthermore, they face challenges related to color variety, functionality, and high sugar content, making them less compatible with the evolving market for health-conscious and functional beverages. Therefore, it is necessary to develop new types of FGR, enrich its color, improve its functionality, and make it an independent beverage.

To address the underutilization of myrtle berries and fermented glutinous rice (FGR) as food ingredients, we proposed a synergistic approach capitalizing on their respective strengths. This study focused on the co-fermentation of glutinous rice and myrtle berries coupled with post-processing techniques to produce a novel myrtle rice beverage (MRB). No literature currently exists on MRB production, suggesting commercial potential for this functional drink combining the delicate floral notes of rice wine with bright, fruity top-notes. Targeting mainstream consumers, this beverage boasts low sugar, mellow alcohol levels, enriched rice and fruit essences, improved nutrition.

2. Materials and methods

2.1. Raw materials and reagents

Ripe myrtle berries and glutinous rice were collected from Wuzhou city, Guangxi province and Huaiyuan county, Anhui Province, China, respectively; Rice leaven was purchased from Angel Yeast Co., LTD; Agar and xanthan gum were purchased from Zhejiang Yinuo Biotechnology Co., LTD; Chromatographic grade methanol was purchased from TEDIA products of U.S.A.; Other analytical reagents were purchased from Sinopharm Group Chemical Reagents Co., LTD.

2.2. The co-fermentation stage of MRB

2.2.1. Co-fermentation process

According to the co-fermentation process of MRB (Figure S1), dried glutinous rice and ripe myrtle berries were selected and pretreated, including cleaning, juicing, steaming, mixing. Then, the rice leaven and water were added, and co-fermentation was carried out under different conditions to obtain the primary fermentation product of MRB, named after myrtle fermented glutinous rice (MFGR).

2.2.2. Optimization of co- fermentation conditions

Uniform design method (Fang & Ma, 2001; Tang & Feng, 2002) was employed to optimize fermentation conditions of MFGR. Based on 100 g glutinous rice, a total of five factors were considered, encompassing myrtle berries content, rice leaven content, fermentation temperature, fermentation time, and water addition amount. An optimization experiment featuring these five factors and eight different levels was conducted by uniform design v5.0, comprehensive evaluations were used as response values, and this evaluation criterion was based on the GB/T15038–2006, titled “General Analysis Method for Wine and Fruit Wine.” The assessment involved the participation of 20 experts who independently assessed and scored the color, sensory attributes, fragrance, and texture of the MFGR. The final score was computed as the average rating given by all 20 experts, with a maximum score of 100.

Data analysis was performed by minitab 14 and matlab 2012b to determine the optimal fermentation conditions. The details of the uniform design table and the comprehensive evaluation criteria are listed in Table 1. Each experimental group underwent parallel testing procedures conducted three times for reliability.

Table 1. The uniform design method was employed to optimize the fermentation conditions of the MFGR, including myrtle berries content, Rice leaven content, fermentation temperature, fermentation time, water addition amount; comprehensive evaluation as a response value, including color, sensory, fragrance, texture.

Test No.	Myrtle berries content (%)	Rice leaven content (%)	Fermentation temperature (°C)	Fermentation time (h)	Water addition amount (%)	Comprehensive evaluation
						Scoring items	Scoring criteria	Score
A B C D E F G H	3% 6% 9% 12% 15% 18% 21% 24%	0.4% 0.6% 0.8% 1% 0.3% 0.5% 0.7% 0.9%	28 36 24 34 26 32 22 30	84 60 96 12 36 72 48 24	100% 90% 80% 70% 60% 50% 40% 30%	Color (25 scores) Sensory (20 scores) Fragrance (25 scores) Texture (30 scores)	Purplish red, transparent Light red, transparent Milky white, opaque	20~25 15~20 <15
							Rice plump, no pollution Rice plump, Surface white mycelium Rice rotten, Surface polychromatic hypha	15~20 10~15 <10
							Intense fruit and rice aromas Light fruit and rice aromas No scent, smelly	20~25 15~20 <15
							Reasonable sweet and sour Light taste Slightly astringent, poor taste	25~30 20~25 <20

2.2.3. Changes of key parameters in co-fermentation process

Key parameters were detected in the co-fermentation process, contributing to the understanding of the basic principles of fermentation. Hence, the fermentation test was performed under the optimized conditions, key parameters were detected at 12 h intervals, including the number of Rhizopus oryzae (Rice leaven mainly contains R. oryzae), total sugar, total acid, and alcohol content in the co-fermentation process. The number of R. oryzae were quantified in accordance with the guidelines outlined in GB4789.2/3/15–2016. The measurement of total sugar, total acid and alcohol content followed the procedures specified in GB/T15038–2006. This comprehensive assessment of the key parameters allowed for a comprehensive understanding of their dynamic evolution during fermentation.

2.3. The post-processing stage of MRB

The processing of MFGR is necessary to improve the taste and expand the potential applications. After being produced under optimal fermentation conditions, the MFGR underwent a series of processing procedures according to the production process of MRB (Figure S1), including grinding, adjustment of sugar–acid ratio and homogenization to MFGR, to produce the final MRB.

2.3.1. Regulate the sugar-to-acid ratio of MFGR

Achieving a balanced sugar-to-acidity ratio is crucial for a pleasant fruit juice flavor. The preferred ratio fruit juices typically ranges from 13:1 to 15:1. In this experiment, the MFGR was ground and filtered using a 120-mesh filter, subsequently, it was diluted in different ratios, and tested for total sugar and acid content. The measurement of total sugar and total acid followed the procedures specified in GB/T15038–2006. The sugar–acid ratio was calculated using formula (1), the ratio range (13:1 ~ 15:1) was used as a standard to determine the optimum MFGR dilution rate. The experiment with each dilution ratio was tested three times (n = 3).

(1) $\mathrm{The}\mathrm{ratio}\mathrm{of}\mathrm{sugar}-\mathrm{to}-\mathrm{acid}=\frac{\mathrm{Total}\mathrm{sugar}}{\mathrm{Total}\mathrm{acid}}$(1)

2.3.2. Homogenization of MFGR

The choice and ratio of stabilizers directly impacts beverage quality and taste. In this study, due to the heat resistance, acid resistance, and cost-effectiveness, xanthan gum and agar were selected as stabilizers for MRB. Combinations of different ratios of agar and xanthan gum (A 0.05%~0.1%, B 0.05%~0.2%, C 0.05%~0.3%, D 0.1%~0.1%, E 0.1%~0.2%, F 0.1%~0.3%) were added to the optimal MFGR dilution, the resulting mixtures were homogenized at 8000 rpm for 5 min (Scientz S10 High Speed Disperser, China), and assessed for the state and rheological characteristics were tested. The stability of the composite system was assessed by clarification column length, and the rheological characteristics were determined using a rheometer (Themo Fisher RS6000, America) with 1 mm plate spacing at 25°C and shear rates from 0 to 100 S^−1.

Subsequently, the beverage was obtained by sterilization at 65°C for 30 m, and the biochemical index was tested, detailed test methods were described in the product characteristics section.

2.4. Product characteristics of MRB

2.4.1. Antioxidant activity of MRB

The antioxidant activity of MRB was determined using DPPH, ABTS, and •OH assay, respectively. The scavenging ability of MRB to DPPH and ABTS was assessed using the following method (Abuelizz et al., 2020), and the scavenging ability for •OH radicals was evaluated using the following method (Moukette et al., 2015), and with slight adjustments. MRB samples at various dilution ratios (1, 1.5, 2, 2.5, 3, and 3.5) were prepared, the absorbance value of sample group, blank group, and control group was designated as A1, A0, A2, respectively. The scavenging rate was calculated according to formula (2), and this process was repeated thrice for each group. VC was used as the positive control, and the results were expressed as $\bar{\text{X}}$ ± S.

(2) $\mathrm{The}\mathrm{scavenging}\mathrm{rate}\left(\mathrm{\%}\right)=\frac{\mathit{A}0-\left(\mathit{A}1-\mathit{A}2\right)}{\mathit{A}0}\times 100$(2)

2.4.2. GC-MS detection of MRB and raw materials

Ripe myrtle berries, FGR), and MRB each possess distinct aromas. As myrtle berries and FGR serve as the raw materials for MRB, it is essential to analyze and compare the volatile components of all three substances to better understand their unique characteristics and the differences among them. Myrtle fruit juice, MRB, and FGR were centrifuged at 12,000 r/min for 10 min, respectively, the three supernatants were extracted, freeze-dried for 12 h, the powder were collected. Three kinds of powder were soaked in chromatographic grade methanol for 2 h, filtered through using 0.22 µm membrane, and centrifuged at 12,000 r/min for 10 min to collect three supernatants. The supernatants were detected by GC-MS (Agilent 7890B-5977A GC-MS hyphenated instrument, America), and 2-octanol as the internal standard. Chromatographic conditions Column: TG-35 ms quartz capillary column (30 m × 0.25 mm, 0.25 µm); Heating procedure: initial temperature 50°C; then rise to 150°C at 10 °C/min, maintain for 2 min; then rise to 200°C at 10 °C/min for 2 min, and then rise to 300°C at 10 °C/min; The temperature of syringe and detector was 280°C, the flow rate of carrier gas (He) was 1 mL/min, and the sample size was 10 µL. Shunt ratio: 1:20.

Mass spectrum conditions Electron bombardment of ion source; Electron energy 70 eV; Ion source temperature 230°C, MS quadrupole rod temperature 150°C; Solvent delay 3 min, quality scanning range m/z 50 ~ 500 (Huang et al., 2023). The corresponding mass spectra of each peak were searched and matched with NIST05 standard mass spectrometry database, and compounds with a matching degree greater than 80% were screened. The peak area normalization method was used for quantitative analysis of components.

2.4.3. Biochemical index of MRB

To obtain the final product, the MRB was sterilized at 65°C for 30 min. Subsequently, the biochemical index of myrtle berries, FGR, MFGR and MRB were analyzed individually. Total sugar, total acid and alcohol content were detected according to the protocols outlined in GB/T15038–2006, titled “General Analytical Methods for Wine and Fruit Wine.” Protein and fat contents were determined in accordance with GB/T5009.5–2008 and GB/T5009.6–2003, respectively. Microbiological assessments encompassed the total colony count, coliform count, and R. oryzae count, which were conducted according to the guidelines specified in GB4789.2/3/15–2016, titled “National Standard for Food Safety Microbial Inspection Total Number of Colonies/Coliform Count.” Each of these parameters was assessed in triplicate, and the results were expressed as $\bar{\text{X}}$ ± S.

3. Results and discussion

3.1. Co-fermentation stage of MRB

3.1.1. Co-fermentation conditions of MFGR

The uniform design method was effectively employed to optimize the co-fermentation conditions of the MFGR, the comprehensive evaluation as the response value. The results were presented in Table 2, and were processed as binary and interaction. The software minitab 14 was used to test the significance of these data and the regression model. The results were shown in Tables 3 and 4, and the binary regression Equation (3)(3) $\begin{array}{rl}& \mathrm{Y}=-618+64.4{\mathrm{X}}_1-2531{\mathrm{X}}_2+34.7{\mathrm{X}}_3+4.53{\mathrm{X}}_4\\ & +0.00461{\mathrm{X}}_5+0.246{{\mathrm{X}}_1}^2+2018{{\mathrm{X}}_2}^2-2.11{\mathrm{X}}_1{\mathrm{X}}_3\end{array}$(3) was obtained.

(3) $\begin{array}{rl}& \mathrm{Y}=-618+64.4{\mathrm{X}}_1-2531{\mathrm{X}}_2+34.7{\mathrm{X}}_3+4.53{\mathrm{X}}_4\\ & +0.00461{\mathrm{X}}_5+0.246{{\mathrm{X}}_1}^2+2018{{\mathrm{X}}_2}^2-2.11{\mathrm{X}}_1{\mathrm{X}}_3\end{array}$(3)

Table 2. The effect of various levels of myrtle berries content, rice leaven content, fermentation temperature, fermentation time, water addition amount on the comprehensive evaluation of MFGR, utilizing the uniform design method to optimize the fermentation conditions. Parallel experiments were conducted three times for each group.

Test No.	Myrtle berries content/X1 (%)	Rice leaven content/X2 (%)	Fermentation temperature/ X3 (°C)	Fermentation time/X4 (h)	Water addition amount/X5 (%)	Color (score)	Sensory (score)	Fragrance (score)	Texture (score)	Comprehensive evaluation (score)
A	3	0.4	28	84	100	12 12 11	15 14 14	17 16 16	22 21 20	66 63 61
B	6	0.6	36	60	90	14 15 13	11 12 10	14 13 12	13 13 12	52 53 47
C	9	0.8	24	96	80	15 16 16	16 15 18	11 12 15	12 11 15	58 58 64
D	12	1	34	12	70	19 17 20	6 5 5	20 18 20	8 5 7	53 45 52
E	15	0.3	26	36	60	23 22 22	18 17 16	18 17 18	12 12 12	71 68 68
F	18	0.5	32	72	50	25 22 23	20 19 20	17 16 16	23 19 22	85 76 81
G	21	0.7	22	48	40	22 20 21	12 13 12	20 22 19	12 11 12	66 66 64
H	24	0.9	30	24	30	13 15 15	11 12 10	18 21 20	11 10 10	53 58 55

Table 3. Significant inspection of regression coefficients.

Independent variable	Coefficient	Standard error of coefficient	T	P
Constant	−617.9	141.2	4.38	0.001
X1	64.39	13.03	4.94	0.000
X2	−2531.4	511.4	4.95	0.000
X3	34.713	7.249	4.79	0.000
X4	4.5293	0.8904	5.09	0.000
X5	0.004608	0.005976	0.77	0.453
X1^2	0.24588	0.06481	3.79	0.002
X2^2	2017.6	412.1	4.90	0.000
X1 X3	−2.1111	0.4455	4.74	0.000

Table 4. Variance analysis of regression equation.

Source of variation	Degree of freedom	Quadratic sum	Mean square	F	p
Regression model	8	2198.63	274.83	27.24	0.000
	15	151.33	10.09	−	−
Total	23	2349.96	−	−	−

Note: Coefficient of determination: R²= = 90.1%.

Analysis of the data presented in Table 3 reveals factor X₅ (p = .453 > 0.05). This suggests that factor ${\mathrm{X}}_5$ had no significant influence on the comprehensive evaluation score, and its coefficient was greater than zero, indicated a positive effect. Therefore, the highest level of X₅, namely, a water addition amount of 100%, should be selected. Conversely, the other four factors demonstrated significant effects (p < .05). and indicated that the myrtle berries content, rice leaven content, fermentation temperature, fermentation time significantly impacted the comprehensive evaluation score of MGFR. Furthermore, the R² (Coefficients of determination for regression models) in Table 4 was 90.1%, indicating that 90.1% of the test data can be explained by this model, which meets the test requirements. P-value of this regression model was zero, signifying its significance.

Utilizing software MatlabR2012b, the binary regression Equation (3)(3) $\begin{array}{rl}& \mathrm{Y}=-618+64.4{\mathrm{X}}_1-2531{\mathrm{X}}_2+34.7{\mathrm{X}}_3+4.53{\mathrm{X}}_4\\ & +0.00461{\mathrm{X}}_5+0.246{{\mathrm{X}}_1}^2+2018{{\mathrm{X}}_2}^2-2.11{\mathrm{X}}_1{\mathrm{X}}_3\end{array}$(3) was solved, resulting in the determination of the optimal fermentation conditions for MFGR production. The optimal conditions (myrtle berries content, rice leaven content, fermentation temperature, fermentation time, water addition amount) for MFGR were 24%, 1%, 22°C, 96 h, 100%, respectively.

In comparison with conventional fermentation conditions of FGR, the MFGR fermentation temperature was lower and the fermentation time was longer. This prolonged fermentation time allows for the complete extraction of various functional components and nutrients from myrtle berries and glutinous rice, maximizing the retention of myrtle berries aroma while preventing degradation at high temperatures. Lower-temperature fermentation reduces the potential for contamination, as it hinders the growth of unwanted bacteria. The slow fermentation process results in thorough metabolic reactions, enhancing the abundance of esters and flavor compounds. In addition, it prevents excessive saccharification and alcoholization, resulting in an elegant and soft final product (López-Malo et al., 2015; Redón et al., 2011). The MFGR produced under these optimal fermentation conditions was characterized by intact rice grains, refreshing sweet and sour flavors, deep red and uniformly clarified juice, and a harmonious blend of rice wine and myrtle berries flavors.

Optimization of MFGR fermentation conditions using the uniform design method allows the attainment of the most suitable conditions for both appearance and taste. Compared to orthogonal design and response surface optimization methods, the uniform design method proves advantageous in handling multifactor and multilevel experiments with fewer trials, a more evenly distributed set of data points, and the ability to rapidly and effectively determine optimal experimental conditions while saving costs and time (Zhou & Jia, 2015).

3.1.2. Changes of key parameters in co-fermentation process

Figure 1 illustrates the changes in R. oryzae count, total sugar, acid, and alcohol content under optimal co-fermentation conditions.

Figure 1. Changes of key parameters in co-fermentation process. The data represent the average of three independent experiments. Error bars represent the standard deviation.

Due to the lower fermentation temperature, R. oryzae propagation rate was initially slow, rapidly increasing after 24 h to 1.22 × 10³ CFU/g at 84 h. As oxygen was depleted and total sugar content sharply rose, mycelia and spores of R. oryzae commenced autolysis, gradually decreasing to 7.2 × 10² CFU/g at 120 h. The total sugar content increased slowly in the first 24 h, primarily due to weak saccharification and its sugar consumption for growth. Total sugar content escalated rapidly after 36 h, reaching 15.92 g/100 g at 96 h. As the count of R. oryzae decreased, the total sugar content tended to stabilize.

R. oryzae perform “sbilateral fermentation”, simultaneously saccharifying and alcoholizing via enzymes including amylase, acid protease, and alcohol dehydrogenases (Zhang et al., 2008). Organic acids like lactic and fumaric acid generated during R. oryzae fermentation primarily serve as the primary source of acidity in fermented glutinous rice (Liu & Jiang, 2001). The total acid increased rapidly after 36 h and reached a maximum at 120 h. R. oryzae can produce alcoholization enzymes that convert fermentable sugars into ethanol. Ethanol adds a mellow flavor to the FGR and is readily absorbed by the human body, promoting blood circulation. Throughout the co-fermentation process, the ethanol content exhibited a gradual increase, reaching 3.2% vol after 36 hours and continued to rise until the end of the fermentation period. The prolonged fermentation time enhanced alcoholization, and the added myrtle berries further promoted alcoholization without saccharification, resulting in relatively high alcohol content of MFGR compared to other FGR productions.

In summary, as co-fermentation time increased, there were positive trends for total sugar, total acid, and alcohol content. However, as the count of R. oryzae decreased and its fermentation abilities weakened, the rate of increase slowed for these parameters. Owing to the high sugar, low pH, and some ethanol generated during MFGR fermentation, the growth and reproduction of mixed bacteria was inhibited without requiring sterilization and aseptic procedures.

3.2. Post-processing of MRB

3.2.1. Regulation of the sugar–acid ratio of MFGR

The characteristics of high sugar, relatively high alcohol content, and rough taste make MFGR unsuitable for regular food. Therefore, further processing is necessary to expand application scenarios. The MFGR was diluted in different ratios and ground, the total sugar and total acid were detected. The sugar–acid ratio was calculated to determine the optimal dilution ratio, and the alcohol content was measured (Figure 2).

Figure 2. Effect of dilution ratio of MFGR on sugar–acid ratio. The data represent the average of three independent experiments. Error bars represent the standard deviation.

From Figure 2, it is evident that as the MFGR was diluted to varying degrees, the sugar–acid ratio exhibited a V-shaped change. The lowest sugar–acid ratio (35:1) was achieved at a dilution rate of 1:3, at this ratio, the amount of citric acid makes it easier to adjust the sugar–acid ratio to the range of 13:1 to 15:1, which is palatable to most people. With increasing dilution, the alcohol content gradually decreased from 2.6 to 0.3%vol. At a dilution rate of 1:3, the alcohol content was 0.6%vol, which is low in alcohol and suitable for consumption by most individuals.

However, after dilution and grinding, the MFGR exhibited instability and stratification. Therefore, it is necessary to add the appropriate stabilizer and high-speed homogenization to improve its stability and taste.

3.2.1. Homogenization of MFGR

Agar and xanthan gum were dissolved in 1:3 ground MFGR, then homogenized at high speed. The compounded systems were allowed to stand for 4 h to observe stratification and assess stability. Rheological characteristics across different stabilizer contents were also analyzed (Figure 3).

Figure 3. Effect of agar and xanthan gum combination on the stability of MRB, (A 0.05%~0.1%, B 0.05%~0.2%, C 0.05%~0.3%, D 0.1%~0.1%, E 0.1%~0.2%, F 0.1%~0.3%).

Figure 3 shows agar and xanthan gum at different proportions noticeably impacted the stability of the MFGR, resulting in varying degrees of liquid stratification. Notably, 0.1% agar and 0.2% xanthan gum best stabilized the system, exhibiting no delamination and 0 cm clarified column. Thus, 0.1% agar and 0.2% xanthan gum were selected as optimal MRB stabilizers.

As seen in Figure 4, increasing shear rate disintegrated or deformed the stabilizers’ assembled molecules, reducing resistance and apparent viscosity of the pseudoplastic fluids. Among the six groups of experiments, MRB with 0.1%-0.2% agar-xanthan gum exhibited the highest viscosity and thickening, aligning with the stability observed in Figure 3.

Figure 4. Effect of agar and xanthan gum combination on rheological properties of MRB.

Agar is a safe, natural polysaccharide thickener, emulsifier, and stabilizer widely used in beverages. Xanthan gum’s double helix structure has strong synergistic effects when mixed with other colloids, networking with small amounts of agar to significantly increase viscosity for thickening and stability (Li et al., 2021). Compounding gels can solve food processing issues, improve quality and safety while reducing additives (Sandhu & Siroha, 2017; Sun et al., 2021).

3.3. Product characteristics of MRB

3.3.1. Antioxidant function of MRB

Figure 5 shows that MRB retained a certain scavenging ability against •OH, ABTS, and DPPH free radicals. Notably, the lower the dilution ratio, the stronger was the scavenging ability, with the highest scavenging rate observed for DPPH. VC, known for its strong antioxidant capacity as a water-soluble vitamin, achieved scavenging rates exceeding 95% for •OH, ABTS, and DPPH free radicals at a concentration of 1.2 mg/mL. While MRB exhibited a weaker antioxidant capacity than VC, the average clearance rate of all three free radicals for MRB remained around 40%.

Figure 5. Antioxidant activity of MRB. The data represent the average of three independent experiments. Error bars represent the standard deviation.

Plant polyphenols are recognized as natural antioxidants with various benefits, including antioxidant, antibacterial, antimutagenic, anti-aging, and anti-inflammatory activities (Re et al., 1999; Yang et al., 2014). Myrtle berries is known to have a much higher polyphenol content than other fruits and grains, and there is a strong correlation between the polyphenol content and antioxidant capacity (Gao et al., 2019).

However, it is important to note that the food processing conditions, light, temperature, pH, and sterilization methods can significantly affect the antioxidant stability of myrtle fruit (Adom & Liu, 2002; Chun et al., 2005; Sakakibara et al., 2003). In this study, the MRB formed after co-fermentation and sterilization retained its ability to scavenge DPPH, ABST, and •OH-free radicals, the myrtle berries overcomes some limitations associated with phenolic compounds, such as degradability and fat solubility, thereby enhancing the bioavailability of MRB. Moreover, the antioxidant properties of MRB reduce the use of preservatives, and making it a healthier and safer beverage.

3.3.2. GC-MS detection of MRB and raw materials

Figure 6a shows that MRB had the highest ester content, followed by alcohols. The most abundant ester was docosadienoic acid methyl ester, making up 28.98% of the relative content. Coming in second was diacetin, a commonly used as a food additive to enhance flavor and quality, at 12.66%, while ethanol constituted 6.89%. Docosadienoic acid methyl ester is a widely utilized unsaturated fatty acid methyl ester. Additionally, MRB also contained small quantities of ketones and organic acids.

Figure 6. (a) GC-MS detection of MRB. (b) GC-MS detection of FGR. (c) GC-MS detection of Myrtle berries. (d) Analysis of the types of volatile organic compound in the three samples.

As shown in Figure 6b, FGR had the highest alcohol content, primarily ethanol and isoamyl alcohol, constituting 11.25% and 7.68% of the relative content, respectively. Additionally, dihydroxyacetone and methylamyl acrylic acid were present at 25.4% and 19.2%, respectively. Notably, dihydroxyacetone, the simplest ketose and a vital intermediate for chemical synthesis, has extensive applications across industries like food, medicine, and cosmetics (Ciriminna et al., 2006; Kim & Hong, 2000).

Figure 6c shows that myrtle berries had the highest dihydroxyacetone content (16.8%), followed by acids and aldehydes, such asbutyric acid(6.56%), palmitic acid(5.12%), lauryl maleic acid(3.19%), hexaldehyde(13%), and hydroxymethylglyo-xal(2.1%). The results were generally aligned with the previous studies (Akyuz et al., 2019; Gorjian et al., 2022), but variations in type and content can be attributed to factors including solvent extraction, fruit ripeness, production area, harvest season, and analytical technique (Aleksic & Knezevic, 2014).

The types of volatile substances in the three samples were classified and analyzed, with results indicating in Figure 6d. MRB had the greatest variety of esters, FGR contained the most alcohol types, and the myrtle berries exhibited the greatest variety of acids. This suggests that during the co-fermentation of myrtle berries and glutinous rice, the alcohol and acid content in the raw materials decreased as they converted into esters. Such transformation intensified flavor and enhanced the nutritional profile of MRB while retaining essential functional components like catechol and caryophyllene from the raw materials. Catechol, commonly found in tea, fruits, and vegetables, has various health benefits, including treating cardiovascular diseases and having antioxidant, antibacterial, and anticancer properties (Patel et al., 2012; Wang et al., 2012). Caryophyllene, primarily found in plants, can help with issues like colitis, alleviate coughs, and combat free radicals (Bouma & Strober, 2003; Cho et al., 2007).

The fermentation can reduce toxicity, enhances biological activity, accelerates nutrient absorption, and improves the taste, It also significantly boosts product flavor and nutritional value, enhancing product competitiveness (Shi et al., 2021). In this study, GC-MS analysis revealed that MRB not only retained the antioxidant components and nutrients from the raw materials while also enriching the aroma.

3.6. Biochemical index of MRB and raw materials

The biochemical index of the myrtle berries, FGR, MFGR and MRB were analyzed, with results were summarized in Table 5. The total sugar and alcohol content of MRB were significantly lower than those in the myrtle berries, FGR, and MFGR, while the acidity of MRB also showed a slight decrease. Most sugars present in FGR and MFGR result from the degradation and transformation of glutinous rice starch by enzymes secreted by R. oryzae. These sugars not only contribute to the sweet taste of foods but also serve as the main energy source for the human body. Certain sugars additionally act as bifidus factors that promoting the growth and reproduction of beneficial intestinal probiotics. Low sugar content is an important characteristics of healthy food feature, and the small amount of ethanol present in MRB can stimulates the esophagus and stomach wall, promoting gastric juice secretion and increasing appetite. Furthermore, compared to other functional beverages MRB contains minor levels of protein and fat, making it suitable for public taste while meeting low sugar requirements for healthy food.

Table 5. Biochemical index of myrtle berries, FGR, MFGR and MRB.

Index	Total sugar(g/100 g)	Alcoholic (%vol)	Total acid(g/100 g)	Protein (%)	Fat (%)	Colony numbers(CFU/g)	Coliform (MPN/g)	R. oryzae numbers(CFU/g)
Description
Myrtle berries	17.5 ± 1.5a	–	0.36 ± 0.5ae	1.5 ± 0.2a	0.35 ± 0.02a	–	–	–
FGR	23.8 ± 4.7b	2.7 ± 0.3a	0.31 ± 0.6 cd	1.8 ± 0.3b	0.42 ± 0.03b	25 ± 1.2a	<1	1.1 × 10^2 ± 1.6a
MFGR	14.6 ± 3.8c	3.2 ± 0.8a	0.34 ± 0.7ac	1.1 ± 0.2c	0.28 ± 0.04c	15 ± 4.2b	<1	1.2 × 10^3 ± 3.9a
MRB	4.1 ± 1.3d	0.6 ± 0.2b	0.28 ± 0.5bd	0.32 ± 0.02d	0.12 ± 0.03d	3.0 ± 1.5c	<1	0.3 ± 0.16b

Note: ”–” no detection.

Mean ± standard deviation (N = 3 trials).

Within a column, different letters indicated significant differences(p < .05), analysis by ANOVA.

The finished MRB product underwent pasteurization and sealing. As a result, the number of colonies, coliform count, and R. oryzae count in the MRB meet the national standards set by the Ministry of Agriculture (NY/T1508–2017) and GB7101–2022 as well as complying with food safety regulations. As FGR and MFGR are produced via R. oryzae fermentation, these products do not necessitate sterilization and may contain a small number of bacteria and residual R. oryzae (Ren & Han, 2012).

4. Conclusions

Myrtle berries boast good medicinal and edible value. A novel type of MRB was produced via the co-fermentation of myrtle berries and glutinous rice using rice wine fermentation techniques. The beverage not only retains the health benefits of FGR, but also incorporates the functional components intrinsic to myrtle berries, making it a healthy drink with a full-bodied flavor, vibrant color, alluring aroma, and enhanced functionality. The development of MRB overcame drawbacks of the individual raw materials, expanded FGR product offerings, and provided a new way for unlocking the potential of myrtle as a valuable resource.

Abbreviations

MRB Myrtle rice beverage; FGR Fermented glutinous rice; MFGR Myrtle-fermented glutinous rice; GC – MS Gas Chromatography-Mass Spectrometer; DPPH 1,1-diphenyl-2-trinitrophenylhydr-azine; ABTS 2, 2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate)

Acknowledgements

We would like to thank Min Han and Jiaxue Qing for their kind support during the sampling process and analysis, and financial support from Dr. Guilan Zhu’s Funding.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

his study were funded by the Project of Natural Science Foundation of Higher Education of Anhui Province (2023AH051295); Blueberry Engineering Technology Research Center of Anhui; Green Food Rural Revitalization Collaborative Technology Service Center of Anhui [GXXT-2022-078]; The Partnership Programme of Hefei Normal University (HXXM2022285; The Project of Natural Science Foundation of Higher Education of Anhui Province</#funding-source; Green Food Rural Revitalization Collaborative Technology Service Center of Anhui.