White birch juice tea beverage as well as preparation method and application thereof

Abstract

The invention provides a white birch juice tea beverage as well as a preparation method and application thereof, and belongs to the technical field of functional beverage preparation. The white birch juice tea beverage disclosed by the invention is prepared from the following components in terms of 100mL: 40 to 50mL of white birch juice, 35 to 45mL of tea leaf extract, 2 to 4g of sugar, 0.15 to 0.25 g of citric acid and the balance of water. Experiments prove that the white birch juice tea beverage prepared by the invention is clear and bright in color and luster, has both the fresh and sweet taste of the white birch juice and the faint scent of the tea leaves, and is harmonious in sour-sweet proportion and fresh and cool in mouth feel. Quality detection results show that the product quality meets national standard requirements.

Description

Technical Field

[0001] This invention belongs to the field of functional beverage technology, specifically relating to a birch sap tea beverage, its preparation method, and its application. Background Technology

[0002] Birch sap, derived from the secondary metabolites of birch trees, is rich in various nutrients such as amino acids, minerals, vitamins, and polysaccharides. It has demonstrated good nutritional and health value in both traditional applications and modern research, and has great application potential.

[0003] The tea beverage market is substantial and diverse, encompassing traditional tea extracts and flavored teas, but suffers from product homogenization and insufficient innovation. Consumer demand for compound beverages that combine tea flavor with the nutritional properties of new ingredients needs further development. However, existing birch sap-related products are mostly single-ingredient extracts or simple blends, with no beverages combining birch sap with tea. On one hand, birch sap itself is easily oxidized and has poor stability, leading to nutrient loss and flavor degradation during processing. On the other hand, when tea extracts (such as tea polyphenols and tea polysaccharides) are blended with birch sap, interactions can cause precipitation, layering, and flavor incompatibility, affecting product quality and shelf life. Existing birch sap product functional claims largely focus on nutritional supplementation, failing to adequately explore the functional synergies of tea beverages and thus not fully meeting consumers' diverse needs for health, function, and taste. Therefore, developing a birch sap tea beverage that solves the technical challenges of blending birch sap with tea, preserves the nutrition and flavor of both, has good stability, a harmonious taste, and unique functions, presents an urgent market demand and technological value. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a birch sap tea beverage, its preparation method and application, wherein the birch sap tea beverage has a clear color, combines the sweetness of birch sap with the fragrance of tea, has a balanced sweet and sour ratio, a refreshing taste, and the product quality meets national standards.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] This invention provides a birch sap tea beverage, which, per 100 mL, comprises the following components: 40-50 mL of birch sap, 35-45 mL of tea extract, 2-4 g of sugar, 0.15-0.25 g of citric acid, and the remainder water.

[0007] Preferably, per 100 mL, it comprises the following components by weight: 42-48 mL of birch sap, 38-42 mL of tea extract, 2.5-3.5 g of sugar, 0.18-0.22 g of citric acid, and the balance being water.

[0008] Preferably, the preparation of the tea extract includes the following steps: mixing tea leaves with water, soaking, filtering, and collecting the filtrate.

[0009] Preferably, the mass-to-volume ratio of tea leaves to water is 1g:40-60mL.

[0010] Preferably, the soaking temperature is 70-90℃ and the soaking time is 15-20 minutes.

[0011] Preferably, the tea leaves include green tea, black tea, or oolong tea.

[0012] The present invention provides a method for preparing the birch sap tea beverage, comprising the following steps: mixing birch sap, tea extract, sugar and citric acid, adjusting the volume with water, homogenizing, sterilizing, and packaging.

[0013] Preferably, the homogenization temperature is 50-70℃, the pressure is 30-40MPa, and the time is 5-15min.

[0014] Preferably, the sterilization temperature is 62-65℃ and the time is 25-35min.

[0015] This invention provides the application of the birch sap tea beverage or the birch sap tea beverage obtained by the preparation method in the preparation of functional beverage products.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention is the first to propose the preparation of birch sap tea beverage by combining birch sap, tea extract, sugar and citric acid in appropriate proportions. The birch sap tea beverage has a clear color, combines the sweetness of birch sap with the fragrance of tea, has a balanced sweet and sour ratio, a refreshing taste, and the product quality meets national standards.

[0018] Furthermore, the birch sap tea beverage preparation process of this invention is simple and easy to implement, and is suitable for large-scale production. Attached Figure Description

[0019] Figure 1 This is a diagram of mouse organ tissues.

[0020] Figure 2 This is a Veen diagram showing the composition of metabolites.

[0021] Figure 3 Principal component analysis of the effects of birch sap on mouse metabolites.

[0022] Figure 4 Partial least squares discriminant analysis was performed to determine the effects of birch sap on mouse metabolites.

[0023] Figure 5 Clustering of differential metabolites in the mouse intestine from birch sap.

[0024] Figure 6 Annotation of KEGG functional pathways for metabolic differentials. The vertical axis represents the secondary classification of KEGG metabolic pathways, and the horizontal axis represents the number of metabolites annotated under that pathway.

[0025] Figure 7 Volcano diagram of differential metabolites in mouse intestine.

[0026] Figure 8 The effect of birch sap addition on sensory scores is shown in the figure. Lowercase letters in the figure indicate within-group significance (p<0.05).

[0027] Figure 9 The graph shows the effect of the amount of tea extract added on sensory scores. Lowercase letters in the graph indicate within-group significance (p<0.05).

[0028] Figure 10 The graph shows the effect of added white sugar on sensory scores. Lowercase letters in the graph indicate within-group significance (p<0.05).

[0029] Figure 11 The graph shows the effect of citric acid addition on sensory scores. Lowercase letters in the graph indicate within-group significance (p<0.05).

[0030] Figure 12 A surface plot and contour plot showing the interaction between the amount of birch sap added and the amount of tea extract added on the sensory score.

[0031] Figure 13 Surface plot and contour plot showing the interaction between the amount of birch sap and the amount of white sugar added on sensory scores.

[0032] Figure 14 Surface plot and contour plot showing the interaction between the amount of birch sap and the amount of citric acid added on sensory scores.

[0033] Figure 15 A surface plot and contour plot showing the interaction between the amount of tea extract and the amount of white sugar added on sensory scores.

[0034] Figure 16 Surface plot and contour plot showing the interaction between the amount of tea extract and the amount of citric acid added on sensory scores.

[0035] Figure 17 Surface plot and contour plot showing the interaction between the amount of added white sugar and the amount of added citric acid on the sensory score. Detailed Implementation

[0036] This invention provides a birch sap tea beverage, comprising, per 100 mL: 40-50 mL birch sap, 35-45 mL tea extract, 2-4 g sugar, 0.15-0.25 g citric acid, and the balance water. Preferably, the birch sap tea beverage comprises, per 100 mL: 42-48 mL birch sap, 38-42 mL tea extract, 2.5-3.5 g sugar, 0.18-0.22 g citric acid, and the balance water; more preferably, comprises, per 100 mL: 44.8 mL birch sap, 40.3 mL tea extract, 3.2 g sugar, 0.21 g citric acid, and the balance water. Unless otherwise specified, the birch sap, sugar, and citric acid in this invention are obtained from commercially available products well-known in the art. The birch sap in this invention is preferably sourced from birch sap collected by Hulunbuir Linhai Forest Management Co., Ltd.

[0037] In this invention, a suitable amount of birch sap and tea extract are combined. At this ratio, the sweet and delicate flavor of the birch sap complements the aroma of the tea extract, ensuring that the tea flavor does not overpower the unique flavor of the birch sap, nor does the birch sap flavor become too prominent and suppress the tea aroma. This ratio allows the characteristics of the birch sap and tea extract to be fully integrated. Adding too much or too little birch sap will disrupt this balance, leading to an unbalanced flavor, unpleasant taste, poor color, and confused aroma, thereby lowering the sensory score.

[0038] In this invention, the appropriate proportion of tea extract added enables the beverage to achieve an optimal balance in terms of flavor, mouthfeel, color, and aroma, thereby obtaining a high sensory score. Conversely, excessive tea extract addition leads to flavor imbalance, bitterness, and a monotonous aroma, while insufficient addition results in a weak flavor, poor color, and inadequate aroma. Therefore, controlling the amount of tea extract added is a key factor in optimizing the sensory quality of birch sap tea beverages.

[0039] In this invention, an appropriate amount of white sugar can enhance the flavor, texture, color, and aroma of the beverage, bringing them to an optimal balance. However, excessive amounts of white sugar can lead to excessive sweetness, a sticky texture, and an unbalanced aroma, while insufficient amounts can result in a beverage with inadequate flavor, a thin texture, and a weak aroma. Therefore, controlling the amount of white sugar added is one of the key factors in optimizing the sensory quality of birch sap tea beverages.

[0040] In this invention, the appropriate amount of citric acid added provides the beverage with moderate acidity, making it refreshing and non-irritating. This acidity harmonizes with the sweetness of birch sap, the richness of tea, and the sweetness of white sugar, creating a balanced taste experience. When the amount of citric acid added exceeds the appropriate level, the sensory score of the tea beverage shows a downward trend. This is because as the amount of citric acid added increases, the acidity of the birch sap tea beverage also increases, causing an imbalance in the sweet and sour ratio and a rough texture.

[0041] In this invention, the preparation of the tea extract includes the following steps: mixing tea leaves with water, soaking, filtering, and collecting the filtrate. The mass-to-volume ratio of tea leaves to water in this invention is 1g:40-60mL, preferably 1g:45-55mL, and more preferably 1g:50mL. The soaking temperature in this invention is 70-90℃ and the soaking time is 15-20min; preferably 75-85℃ and the soaking time is 16-19min; more preferably 80℃ and the soaking time is 18min. The tea leaves in this invention include green tea, black tea, or oolong tea, preferably green tea, and more preferably Biluochun, Maojian, or Longjing. The Biluochun tea in this invention is preferably sourced from Nanjing Bixuan Cultural Communication Co., Ltd.

[0042] This invention also provides a method for preparing the birch sap tea beverage, comprising the following steps: mixing birch sap, tea extract, sugar, and citric acid; adjusting the volume with water; homogenizing; sterilizing; and packaging. The birch sap tea beverage prepared by this invention has a clear color, combining the sweetness of birch sap with the aroma of tea, a balanced sweet and sour ratio, and a refreshing taste.

[0043] The homogenization temperature of this invention is 50-70℃, the pressure is 30-40MPa, and the time is 5-15min; preferably, the homogenization temperature is 55-65℃, the pressure is 32-38MPa, and the time is 6-14min; more preferably, the homogenization temperature is 60℃, the pressure is 35MPa, and the time is 10min. This invention involves slowly pouring the prepared and shaken birch sap tea beverage into a homogenizer for homogenization, making the particles in the beverage smaller and more evenly distributed, thus improving product stability and taste. The sterilization temperature of this invention is 62-65℃, and the time is 25-35min; preferably, the sterilization temperature is 63-64℃, and the time is 28-32min; more preferably, the sterilization temperature is 63.5℃, and the time is 30min. This invention kills harmful microorganisms in the beverage, such as bacteria, molds, and yeasts, ensuring the microbiological safety and shelf life of the product.

[0044] This invention provides the application of the birch sap tea beverage, or the birch sap tea beverage obtained by the preparation method, in the preparation of functional beverage products. The birch sap tea beverage of this invention retains the original functions of birch sap.

[0045] In this invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0046] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0047] The raw materials used in the following examples are as follows:

[0048] Biluochun Tea: Selected high-quality tea leaves, ensuring no impurities or mold. Source: Nanjing Bixuan Cultural Communication Co., Ltd. Birch Sap: Fresh birch sap, requiring no spoilage or pollution, freshly collected sap stored at low temperatures, sourced from birch trees collected by Hulunbuir Linhai Forest Management Co., Ltd. White Sugar: Source: Inner Mongolia Dajiang Sugar Industry Co., Ltd., 500g / bag. Citric Acid (Food Grade): Source: Shandong Yingxuan Industrial Co., Ltd., 500g / bag.

[0049] Example 1

[0050] A birch sap tea beverage, per 100 mL, is prepared from the following components: 44.8 mL of birch sap, 40.3 mL of tea extract, 3.2 g of white sugar, 0.21 g of citric acid, and the remainder water.

[0051] A method for preparing a birch sap tea beverage is as follows:

[0052] (1) Weigh out Biluochun tea leaves and place them in a clean beaker according to a mass-volume ratio of 1g:50mL. Pour in drinking water and soak at 85℃ for 15 minutes while stirring. After soaking, filter the tea leaves using a filter screen to separate the tea residue from the extract. Keep the tea extract for later use.

[0053] (2) Preparation of citric acid solution: Citric acid and water are mixed at a mass-volume ratio of 1g:40mL to obtain citric acid solution.

[0054] (3) Slowly add the birch sap to the tea extract according to the above formula, stirring constantly to ensure initial mixing. Monitor the temperature of the mixture using a thermometer and adjust it to approximately 20°C in a cold water bath. Then add the granulated sugar and continue stirring until completely dissolved to obtain the mixture. Slowly pour the prepared citric acid solution into the mixture while continuously stirring to ensure uniform mixing. Finally, transfer the mixture to a 1L volumetric flask and dilute to 1L with distilled water. Gently shake well after dilution.

[0055] (4) Turn on the homogenizer and preheat it to 60°C. Set the homogenization pressure to 35MPa and the time to 10min. Slowly pour the prepared and shaken birch sap tea beverage into the homogenizer for homogenization.

[0056] (5) Transfer the homogenized birch sap tea beverage to a sterilization device and sterilize at 65°C for 30 minutes.

[0057] (6) After sterilization, wait for the beverage to cool to a suitable temperature (usually room temperature), and fill the beverage into sterilized packaging containers in a sterile environment. Store the product in a cool, dry, and ventilated place, avoiding direct sunlight and high temperatures to prevent quality degradation.

[0058] Example 2

[0059] A birch sap tea beverage, per 100 mL, is prepared from the following components in parts by weight: 45 mL birch sap, 35 mL tea extract, 2 g white sugar, 0.2 g citric acid, and the remainder water.

[0060] The preparation method is the same as in Example 1.

[0061] Example 3

[0062] A birch sap tea beverage, per 100 mL, is prepared from the following components in parts by part: 45 mL of birch sap, 40 mL of tea extract, 3 g of white sugar, 0.15 g of citric acid, and the remainder water.

[0063] The preparation method is the same as in Example 1.

[0064] Example 4

[0065] A birch sap tea beverage, per 100 mL, is prepared from the following components in parts by part: 45 mL of birch sap, 40 mL of tea extract, 3 g of white sugar, 0.2 g of citric acid, and the remainder water.

[0066] The preparation method is the same as in Example 1.

[0067] Example 5

[0068] A birch sap tea beverage, per 100 mL, is prepared from the following components in parts by part: 45 mL of birch sap, 40 mL of tea extract, 2 g of white sugar, 0.2 g of citric acid, and the remainder water.

[0069] The preparation method is the same as in Example 1.

[0070] Experimental Example 1: Safety and Functional Evaluation of Birch Sap

[0071] 1. Experimental Animals and Materials: Kunming mice (Henan Skebest Biotechnology Co., Ltd.), weighing 18–22 g, six weeks old, female. All experimental mice were provided with free access to food and water during the rearing period, and their sterile food, drinking water, and bedding were changed daily. The animal room was kept under 12-hour light-dark cycles, and the temperature was maintained at 23±2℃. The mice underwent a 7-day acclimatization period before the start of this experiment.

[0072] Experimental material: birch sap, which was collected from birch trees by Hulunbuir Forest Management Co., Ltd.

[0073] 2. Test methods

[0074] 2.1 Safety evaluation of birch sap

[0075] The experiment was conducted according to the People's Republic of China National Standard GB / T23179-2008 "Subacute Toxicity Test for Feed Toxicology Evaluation". Mice were randomly divided into two groups of 15 each: a control group (0.9% saline, group C) 20 mL / kg / day and an experimental group (birch sap, group T) 20 mL / kg / day. Mice were administered the medication once daily by gavage for 14 consecutive days. During the experiment, the mice's growth, symptoms of poisoning, excretion, and mortality were observed. Mouse weight was recorded before each day's gavage. After 14 days, the mice were fasted for 6 hours prior to the experiment, and blood was collected using ocular sampling. Blood parameters were analyzed, including complete blood count (white blood cells, red blood cells, hemoglobin, platelets, lymphocytes, neutrophil ratio, and monocyte ratio). The required organs (liver, spleen, and lungs) were dissected, and their mass (mg) was measured and calculated. The results were compared with the control group.

[0076] Organ index = Organ mass (g) / Body mass (g)

[0077] 2.2 Study on the functional properties of birch sap

[0078] 2.2.1 Effects on animal growth performance

[0079] Twelve healthy 6-week-old female Kunming mice were randomly divided into two groups. In addition to receiving a standard diet, group C was administered 20 mL / kg / day of 0.9% saline via gavage, while group T was administered 20 mL / kg / day of birch sap via gavage for 28 consecutive days. Gavage was performed daily at 9:00 AM. At the start of the experiment, mice were weighed every seven days on an empty stomach.

[0080] 2.2.2 Testing of anti-fatigue functional properties and determination of biochemical indicators

[0081] On day 29 after gavage, mice were subjected to a swimming test 30 minutes after gavage. Before the swimming test, the mice were weighed and placed in a temperature-controlled swimming incubator according to their groups. The water temperature was set at 25±2℃, and the test was conducted once daily for 20 minutes for 7 consecutive days. Intensive swimming training was then provided for 3 days, twice daily with a 3-hour interval. Throughout the swimming process, the mice's movement was continuously observed, and a glass rod was used to agitate their limbs. A mouse was considered exhausted if its head remained submerged for more than 10 seconds. After the swimming test, the mice were immediately removed, dried with a towel, and returned to their cages. Then, blood was collected from the mice's eyes, and the levels of biochemical indicators (blood lactate, blood urea nitrogen, and liver glycogen) were measured. The detailed steps for the detection of each indicator were performed in accordance with the instructions of the mouse liver glycogen ELISA kit (Shanghai Keshun Biotechnology Co., Ltd.), blood urea nitrogen (UV-glutamate dehydrogenase method) kit (Shanghai Keshun Biotechnology Co., Ltd.), and blood lactate (LD) test kit (Nanjing Jiancheng).

[0082] 2.2.3 Effects on mouse organs

[0083] After blood collection as described in section 2.2.2, mice were euthanized by cervical dislocation. Major organs such as the liver, spleen, and lungs were quickly harvested. After rinsing with sterile physiological saline (0.9%) to remove residual blood, the samples were blotted dry with filter paper. Tissue samples were fixed with 4% paraformaldehyde and processed according to standard pathological procedures: sequential dehydration with graded ethanol, clearing with xylene, and paraffin embedding. 5μm sections were prepared, stained with hematoxylin and eosin (HE), mounted with neutral resin, and the morphological changes were observed under an optical microscope.

[0084] 2.2.4 Effects on animal immune indicators

[0085] Animal immune indicators were measured in the blood samples collected from mice in section 2.2.2.

[0086] Serum complement C3 and immunoglobulin (IgG, IgA, IgM) levels were measured strictly according to the operating procedures of commercially available ELISA kits. Complement C3 was detected using a mouse complement protein 3 (C3) ELISA kit, IgA was detected using a mouse immunoglobulin A (IgA) ELISA kit, IgM was detected using a mouse immunoglobulin M (IgM) ELISA kit, and IgG was detected using a mouse immunoglobulin G (IgG) ELISA kit. All kits were obtained from Shanghai Keshun Biotechnology Co., Ltd.

[0087] All assays included a standard gradient (0-100% binding rate) and double-duplicate controls. Absorbance was measured at 450 nm using a microplate reader, and sample concentrations were calculated by fitting a four-parameter logistic curve. The entire experiment adhered to the quality control requirements outlined in the kit instructions, with intra-batch coefficients of variation all <10%, ensuring the accuracy and reproducibility of the results.

[0088] 2.2.5 Non-targeted metabolomics analysis of animal feces

[0089] 2.2.5.1 Sample Preparation

[0090] Take 50 mg of fecal sample into a 2 mL centrifuge tube and add a 6 mm diameter grinding bead. Extract metabolites using 400 μL of extraction buffer (methanol:water = 4:1 (v:v)) containing 0.02 mg / mL internal standard (L-2-chlorophenylalanine). Grind the sample solution in a cryo-tissue homogenizer for 6 min (-10℃, 50 Hz), followed by cryogenic extraction for 30 min (5℃, 40 kHz). Incubate the sample at -20℃ for 30 min, centrifuge for 15 min (4℃, 13000 g), and transfer the supernatant to a vial with an inner tube for analysis.

[0091] 2.2.5.2 LC-MS / MS Analysis

[0092] The samples were analyzed by LC-MS / MS using a Thermo Fisher Scientific UHPLC-Q Exactive HF-X system.

[0093] 2.2.5.3 Substance Identification and Analysis

[0094] After the LC-MS was completed, the raw LC-MS data were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Finally, a data matrix of retention time, mass-to-charge ratio, and peak intensity was obtained. At the same time, the MS and MSMS mass spectrometry information was matched with the public metabolic databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) as well as a self-built library to obtain metabolite information.

[0095] The data matrix after the database search was analyzed. First, the data matrix was preprocessed as follows: Missing values ​​were removed using the 80% rule, meaning variables with more than 80% non-zero values ​​in at least one sample group were retained. Then, missing values ​​were filled (the minimum value in the original matrix was used to fill the gaps). To reduce errors caused by sample preparation and instrument instability, the response intensity of the sample mass spectrometry peaks was normalized using the summation normalization method, resulting in a normalized data matrix. Simultaneously, variables with a relative standard deviation (RSD) > 30% for QC samples were removed, and logarithmic transformation (log10) was performed to obtain the final data matrix used for subsequent analysis.

[0096] 2.2.5.4 Data Processing

[0097] Secondly, the preprocessed data matrix was analyzed using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) using the ropls package in R (Version 1.6.2), and the model stability was evaluated using seven-cycle cross-validation. The selection of significantly differentially expressed metabolites was based on the variable weights (VIPs) obtained from the OPLS-DA model and the p-values ​​of Student's t-test; metabolites with VIP > 1 and p < 0.05 were considered significantly differentially expressed. Differentially expressed metabolites were annotated using the KEGG database (https: / / www.kegg.jp / kegg / pathway.html) to identify the pathways involved. Pathway enrichment analysis was performed using the scipy.stats package in Python, and Fisher's exact test was used to identify the biological pathways most relevant to the experimental treatment.

[0098] 3. Results Analysis

[0099] 3.1 Safety evaluation of birch sap

[0100] 3.1.1 Oral gavage animal pathogenicity test

[0101] During the 14-day observation period, mice that underwent a single oral gavage of birch sap exhibited good physiological and mental condition. Their fur was smooth and clean, they moved agilely and actively, and their food and water intake remained normal. No adverse reactions such as diarrhea, vomiting, or tremors were observed, nor were any poisoning-related symptoms or abnormal behaviors detected. These results indicate that under the experimental conditions, birch sap has good safety for mice and did not exhibit acute toxicity. This safety assessment provides important evidence for the further development and application of birch sap.

[0102] 3.1.2 Effects of birch sap on animal blood parameters

[0103] Table 1 shows that after gavage administration of birch sap, no toxicologically significant abnormalities were observed in the blood routine indicators of mice in the experimental group (T group) compared to the control group (C group). All parameters were within the normal physiological range, and there were no significant differences between the groups (p>0.05). This indicates that birch sap did not cause significant hematologic toxicity. (T group: 3.038±0.78×10⁻⁶) 9 The white blood cell count ( / L) was slightly higher than that in group C (2.02±1.14×10⁻⁶). 9 The birch sap concentration was 8.838 ± 0.60 × 10⁻⁶ L, but still within the normal range, and there was no significant difference (p > 0.05). This indicates that birch sap did not induce inflammation or excessive activation of the immune system. Group C (8.838 ± 0.60 × 10⁻⁶ L) 12 / L) Red blood cells and T group (9.132±1.88×10 12 The hemoglobin content in group C (153.96±5.86 g / L) was similar to that in group T (156.48±6.45 g / L), and both were within normal physiological levels, indicating that birch sap does not affect hematopoietic function or red blood cell metabolism. Group C (851.67±17.99×10⁻⁶ g / L) had a similar hemoglobin content to group T (156.48±6.45 g / L), which is within normal physiological levels, indicating that birch sap does not affect hematopoietic function or red blood cell metabolism. 9 Platelet count ( / L) was compared with that in group T (863.03±25.49×10⁻⁶). 9 Compared with the previous group ( / L), there was no significant difference between the groups (p>0.05). Group T (2.30±0.57×10) 9 The lymphocyte count was slightly higher than that in group C (1.58 ± 0.85 × 10⁹ / L), but not as high as in group C (1.58 ± 0.85 × 10⁹ / L). 9 The lack of significant difference in per liter ( / L) indicates that the immune system was not abnormally stimulated. Combined with the following experimental data, it can be demonstrated that birch sap has reliable safety at the tested dose and did not have adverse effects on the hematopoietic or immune systems.

[0104] Table 1. Detection of routine blood parameters in mice

[0105] index Group C Group T Reference range <![CDATA[White blood cells (10 9 / L)]]> 2.02±1.14 3.038±0.78 0.80-10.60 <![CDATA[Red blood cells (10 12 / L)]]> 8.838±0.60 9.132±1.88 6.50-11.50 Hemoglobin (g / L) 153.96±5.86 156.48±6.45 110-165 <![CDATA[Platelets (10 9 / L)]]> 851.67±17.99 863.03±25.49 400-1600 <![CDATA[Lymphocytes (10 9 / L)]]> 1.58±0.85 2.30±0.57 0.60-8.90 Neutrophil percentage (%) 12.28±2.87 11.00±1.98 6.5-50.0 Monocyte percentage (%) 6.82±3.83 9.646±4.41 0.9-18.0

[0106] Note: For intergroup comparisons, the absence of a subscript indicates no significant difference (p>0.05), while different lowercase letters indicate significant differences (p<0.05).

[0107] 3.1.3 Effects of birch sap on organ indices in mice

[0108] In animals and humans, abnormal nutritional status or visceral lesions can lead to abnormal changes in certain physiological indicators, including changes in organ weight. Therefore, observing changes in organ weight can provide insight into the body's nutritional status and the health of internal organs. The spleen and liver, in particular, are considered the most basic and routine indicators. Table 2 shows the effects of birch sap administered orally on organ indices in mice. The results indicate that, compared to the control group, the spleen index in group T (0.10±0.02) was slightly lower in group T than in group C (0.12±0.04), but the difference was not significant, suggesting that birch sap did not cause abnormal proliferation or atrophy of immune organs, indicating no risk of immunotoxicity. The liver index in group T (4.80±0.36) was similar to that in group C (4.98±0.46), with no significant difference, indicating that birch sap did not increase the metabolic load on the liver or cause liver damage (such as inflammation or fatty degeneration). The lung index values ​​of the two groups were almost identical (Group C 0.22±0.02, Group T 0.21±0.01), further ruling out the potential toxicity of birch sap to the respiratory system. The differences were not significant (P>0.05), indicating that birch sap had no effect on the vital organs of mice and possessed good safety.

[0109] Table 2. Effects of organ indices on mice in each group.

[0110] Group Spleen Index Liver index Lung Index Group C 0.12±0.04 4.98±0.46 0.22±0.02 Group T <![CDATA[0.10±0.02 a ]]> 4.80±0.36 0.21±0.01

[0111] Note: For intergroup comparisons, data without subscripts indicate no significant difference (p>0.05), while different lowercase letters indicate significant differences (p<0.05).

[0112] 3.2 Functional Characteristics

[0113] 3.2.1 Effects of birch sap on animal growth performance

[0114] Changes in mouse body weight are an important indicator of the effect of birch sap on their growth performance. Table 3 shows the changes in mouse body weight after 28 days of oral gavage with birch sap. Initial body weight (day 0): There was no significant difference between the two groups (Group C 18.71±0.69g, Group T 18.91±0.87g) (p>0.05), indicating consistent starting conditions. On day 7, Group T (30.14±2.07g) was significantly higher than Group C (27.62±1.20g) (p<0.05), indicating that birch sap promoted early body weight gain. On day 14, group T (35.12±1.47g) was still significantly higher than group C (32.19±2.27g) (p<0.05). On day 21, group T (38.28±1.29g) was still significantly higher than group C (35.54±1.48g) (p<0.05). On day 28, group T (40.80±1.65g) was still significantly higher than group C (38.37±1.20g) (p<0.05). This indicates that birch sap has a sustained positive effect on long-term growth performance, and the effect is safe and continuous, with potential growth regulation function.

[0115] Table 3. Changes in mouse body weight in each group

[0116] Group 0d 7d 14d 21d 28d Group C (g) <![CDATA[18.71±0.69 a ]]> <![CDATA[27.62±1.20 a ]]> <![CDATA[32.19±2.27 a ]]> <![CDATA[35.54±1.48 a ]]> <![CDATA[38.37±1.20 a ]]> Group T (g) <![CDATA[18.91±0.87 a ]]> <![CDATA[30.14±2.07 b ]]> <![CDATA[35.12±1.47 b ]]> <![CDATA[38.28±1.29 b ]]> <![CDATA[40.80±1.65 b ]]>

[0117] Note: For intergroup comparisons, data with the same lowercase letter in the data headings or no headings indicate no significant difference (p>0.05), while different lowercase letters indicate significant difference (p<0.05).

[0118] 3.2.2 Testing of the anti-fatigue properties and determination of biochemical indicators of birch sap

[0119] After the exhaustive swimming test, the serum biochemical indicators of mice are shown in Table 4. All indicators showed statistically significant differences between the experimental and control groups (p<0.05), and met the effective improvement threshold of exercise physiology. The liver glycogen content in group T (48.84±1.35 mg / g) was significantly higher than that in group C (26.26±2.10 mg / g), indicating that birch sap can promote liver glycogen storage, provide more energy substrates for exercise, and delay the onset of fatigue. The urea nitrogen content in group T (25.41±9.16 mmol / L) was significantly lower than that in group C (36.19±8.57 mmol / L), reflecting a reduction in protein catabolism, suggesting that birch sap may alleviate exercise-induced muscle damage and reduce the accumulation of fatigue-related metabolites. The blood lactate level in group T (2.29±1.57 mmol / L) was significantly lower than that in group C (5.03±1.49 mmol / L), suggesting that birch sap can improve anaerobic metabolism efficiency and reduce blood lactate accumulation, thereby alleviating muscle soreness and fatigue after exercise. In conclusion, birch sap exhibits significant anti-fatigue properties by regulating energy metabolism and reducing the accumulation of fatigue products.

[0120] Table 4. Detection of serum biochemical indicators in mice

[0121] index Group C Group T liver glycogen <![CDATA[26.26±2.10 a ]]> <![CDATA[48.84±1.35 b ]]> urea nitrogen <![CDATA[36.19±8.57 a ]]> <![CDATA[25.41±9.16 b ]]> Blood lactate <![CDATA[5.03±1.49 a ]]> <![CDATA[2.29±1.57 b ]]>

[0122] Note: For intergroup comparisons, data with the same lowercase letter or no lowercase letter in the data label indicate no significant difference (p>0.05), while different lowercase letters indicate significant differences (p<0.05).

[0123] 3.2.3 Effects of birch sap on mouse organs

[0124] At the end of the experiment, the mice were dissected, and histological examinations were performed on their major internal organs, including the liver, lungs, and spleen. Figure 1 As shown, the liver, lung, and spleen structures of mice in the control group (Group C) and the experimental group (Group T) were observed using H&E staining. In the lung tissue, the alveolar structure was clear, the alveolar septa were of uniform thickness, and there was no congestion, edema, or inflammatory exudation; the bronchial epithelial cells were neatly arranged. In the liver tissue, the hepatic lobule structure was intact, the hepatocyte cords were neatly arranged, and no fatty degeneration, inflammatory infiltration, or fibrotic lesions were observed; the central vein and portal area structures were normal. In the spleen tissue, the white pulp and red pulp were clearly demarcated, the lymphoid follicle structure was normal, and no abnormal proliferation or fibrosis was observed. The organ tissue structures in both groups maintained normal morphological characteristics, and no pathological changes (such as necrosis, inflammation, fibrosis, etc.) were observed. This verifies the safety of birch sap for consumption; at the experimental dose, it has no toxic effects on major organs, and the risk of long-term consumption is low.

[0125] 3.2.4 Effects of birch sap on animal immune indicators

[0126] Table 5 shows that after gavage administration of birch sap, the immune indicators of mice in group T were significantly higher than those in the control group (group C) (p<0.05), indicating that birch sap has a clear immunomodulatory function. The complement C3 content in group T (168.10±6.16 μg / mL) was significantly higher than that in group C (76.84±9.57 μg / mL). This activation of the complement system and enhanced pathogen clearance ability indicates that birch sap can enhance innate immune defense. The immunoglobulin IgG content in group T (26.69±2.07 μg / mL) was significantly higher than that in group C (15.10±2.11 μg / mL), reflecting enhanced humoral immunity and improved antibody-mediated adaptive immune response. The IgM content in group T (4645.21±342.20 μg / mL) was significantly higher than that in group C (2532.11±218.87 μg / mL). The elevated antibody levels in the early stages of acute infection indicate an enhanced rapid immune response. The IgA content in group T (329.30±31.89 μg / mL) was significantly higher than that in group C (176.50±22.96 μg / mL), indicating enhanced mucosal immunity and increased defense potential against respiratory or gastrointestinal infections.

[0127] In conclusion, birch sap can significantly activate the complement system and increase the levels of multiple types of immunoglobulins, systematically enhancing the immune function of mice, and its effects are safe and reliable.

[0128] Table 5 Detection of immune indicators in mice

[0129]

[0130]

[0131] Note: For intergroup comparisons, data with the same lowercase letter in the data headings or no headings indicate no significant difference (p>0.05), while different lowercase letters indicate significant difference (p<0.05).

[0132] 3.3 Animal Non-Targeted Omics Metabolic Analysis

[0133] 3.3.1 Metabolite Composition Analysis

[0134] The number of common and unique metabolites was statistically analyzed using Veen analysis. For example... Figure 2 As shown, group C has 1642 metabolites, of which 5 are unique metabolites; group T has 1651 metabolites, of which 14 are unique metabolites. Groups C and T together contain 1637 compounds.

[0135] The classification of specific metabolites using HMDB subclasses revealed in Table 6 that metabolites specific to group C are mainly associated with bile acid metabolism and steroidal compounds (such as bile acids and ergot alkaloids), potentially involving liver function or digestive metabolism. In contrast, metabolites specific to group T exhibit broader biological activity, including neurotransmitter analogs (indole carboxylic acids), antioxidants (isoflavones), steroidal compounds, and peptides, involving metabolic activities of the nervous, endocrine, and immune systems.

[0136] Table 6. Composition of mouse intestinal metabolites

[0137]

[0138] Note: C is the control group, and T is the experimental group.

[0139] 3.3.2 Principal component analysis of metabolites

[0140] Principal component analysis (PCA), as an unsupervised pattern recognition method, can extract the principal components with the most information through data dimensionality reduction, thereby determining the linear or nonlinear structure of the data and intuitively reflecting the differences in metabolites between samples. For example... Figure 3 As shown, PC1 and PC2 explained 32.00% and 15.60% of the data, respectively, demonstrating high explanatory power. No significant separation trend was observed between groups C and T, but the Adonis intergroup difference test (P < 0.05) indicated a significant difference between the two groups.

[0141] 3.3.3 Partial Least Squares Discriminant Analysis of Metabolites

[0142] Partial least squares discriminant analysis (PLS-DA) can maximize the differences between groups according to a predefined classification (Y variable), which is beneficial for finding differentially expressed metabolites and achieving better separation results than PCA. Figure 4 As shown, the explanatory values ​​of PC1 and PC2 were 29.90% and 10.60%, respectively. Groups C and T exhibited a clear separation trend under the PLS-DA model, and the Adonis intergroup difference test p<0.05, indicating a significant difference between the two groups. This suggests that birch sap has a significant effect on intestinal metabolites in mice.

[0143] 3.3.4 Differential Metabolite Cluster Analysis

[0144] The Euclidean algorithm was used to perform sample-level clustering of the top 30 differentially abundant metabolites, such as... Figure 5 As shown, 18 differential metabolites were found in relatively higher amounts in group T, and 12 were found in relatively higher amounts in group C.

[0145] Classification using the HMDB database revealed that aromatic amino acids and their derivatives (L-Phenylalanine, L-Tyrosine, L-Tryptophan, Indole-3-Lactic Acid, N-Methyl-D-Aspartic Acid), which are relatively abundant in group T, affect neurotransmitter synthesis and metabolic regulation; biogenic amines and neurotransmitter-related substances (Octopamine, Piperidine, Indoline) may be involved in stress response and energy metabolism; dipeptides and small peptides (Phe-Pro, Phe Leu, Leucylproline, Ile Gln, Acetyl-DL-Valine) are mainly involved in protein degradation and amino acid metabolism; phenylpropanoids and phenolic metabolites (2-Hydroxycinnamic Acid, Riesling Acetal) have antioxidant and anti-inflammatory effects; and other organic acids and their derivatives (Nopalinic Acid, Pro PheAcetyl-DL-Valine) may play a role in energy metabolism and microbial metabolism. Nucleosides and their analogues (Sapacitabine) with relatively high T-group content have antitumor activity; peptide compounds (Ile-Ile-Ile-Pro, Ulimorelin) have the effects of promoting appetite and regulating metabolism; fatty acids and their esters (Behenic Acid, Arachidic Acid) may affect lipid metabolism and energy storage. Alkaloids and their derivatives (Dapta, Blumealactone B, Austinoneol, Deltaline) may have neuroprotective, immunomodulatory, and antioxidant effects; steroids and glycosides (2-Phenylethyl Beta-D-Glucopyranoside) have antioxidant and antibacterial activities; nitrogen-containing heterocyclic compounds (Ethyl 3-((6-(4,5-Dihydro-1H-Benzo[D]Azepin-3(2H)-Yl)-2-(Pyridin-2-Yl)Pyrimidin-4-Yl)Amino)Propanoate) may have anti-inflammatory and neuroactive effects. In conclusion, feeding with birch sap may reduce lipid metabolism, but it increases amino acid metabolism in mice, affecting the nervous system and protein synthesis.

[0146] 3.3.5 Statistical analysis of the KEGG functional pathway of differential metabolites

[0147] Statistical mapping was performed using the KEGG Pathway database to identify the metabolic pathways (tertiary classification) to which differentially metabolized substances belong. For example... Figure 6As shown, among the 227 differentially metabolites, 195 were related to metabolic pathways, 12 to organismal system pathways, 7 to human disease pathways, 7 to genetic information processing pathways, and 6 to environmental information processing pathways. These results indicate that birch sap has a wide-ranging impact on mouse metabolism, primarily involving the regulation of metabolic pathways.

[0148] 3.3.6 Analysis of significantly different metabolites

[0149] The screening criteria for differentially expressed metabolites were based on the student's t-test (two-tailed test, P-value < 0.05), VIP-pred-OPLS-DA > 1, and a fold change in up / down regulation of 1. Figure 7 As shown, among the 1621 metabolites in groups T and C, 190 significantly upregulated metabolites, 37 significantly downregulated metabolites, and 1349 metabolites with no significant difference were annotated.

[0150] Experiment Example 2: Single-factor experiment on the preparation of birch sap tea beverage

[0151] The preparation method of the birch sap tea beverage involved in this experimental example is the same as that in Example 1, except for the following specified parameters.

[0152] 1. Experimental Design

[0153] (1) Optimization of birch sap addition amount

[0154] Under the conditions of fixed addition of tea extract (35% v / v), white sugar (2% g / mL), and citric acid (0.2% g / mL), the effects of birch sap addition (v / v) of 35%, 40%, 45%, 50%, and 55% on the sensory quality of tea beverages were investigated.

[0155] (2) Optimization of tea extract addition amount

[0156] Under the conditions of fixed addition of birch sap (45% v / v), white sugar (2% g / mL), and citric acid (0.2% g / mL), the effects of tea extract addition (v / v) of 25%, 30%, 35%, 40%, and 45% on the sensory quality of tea beverages were studied.

[0157] (3) Optimization of white sugar addition amount

[0158] Under the conditions of fixed addition of birch sap (45% v / v), tea extract (40% v / v), and citric acid (0.2% g / mL), the effects of added white sugar (g / mL) of 1%, 2%, 3%, 4%, and 5% on the sensory quality of tea beverages were analyzed.

[0159] (4) Optimization of citric acid addition

[0160] Under the conditions of fixed addition of birch sap (45% v / v), tea extract (40% v / v), and white sugar (3% g / mL), the effects of citric acid addition (g / mL) of 0.05%, 0.1%, 0.15%, 0.2%, and 0.25% on the sensory quality of tea beverages were investigated.

[0161] 2. Sensory evaluation

[0162] This experiment followed the sensory evaluation methods specified in GB / T 21733-2008 "Tea Beverages". A panel of 10 professionally trained sensory evaluators independently scored the texture, flavor characteristics, and appearance of the birch sap tea. Participants were prohibited from communicating during the evaluation process and were required to rinse their mouths with water before and after each tasting. The sensory evaluation indicators and scoring criteria are shown in Table 7 below.

[0163] Table 7 Sensory Evaluation Standards for Birch Sap Tea Beverage

[0164]

[0165] 3. Results Analysis

[0166] (1) Effect of birch sap addition on sensory scores of tea beverages

[0167] The effect of different amounts of birch sap added on the sensory scores of birch sap tea beverages, such as Figure 8 As shown, different amounts of birch sap were mixed with 35% tea extract, 2% white sugar, and 0.2% citric acid. The optimal blending ratio for the birch sap tea beverage was determined based on sensory evaluation. The highest sensory score was achieved when the birch sap content was 45%. At this ratio, the sweet and delicate flavor of the birch sap complemented the aroma of the tea extract, ensuring that the tea flavor did not overpower the unique flavor of the birch sap, nor did the birch sap flavor become too prominent and suppress the tea aroma. This ratio allowed the characteristics of the birch sap and tea extract to fully blend. Adding too much or too little birch sap would disrupt this balance, leading to flavor imbalance, unpleasant taste, poor color, and chaotic aroma, thus lowering the sensory score. When the amount of birch sap added was 45%, the sensory score was the highest, the aroma of birch sap was more harmonious, and the color was more uniform. Therefore, three levels of birch sap addition were selected: 40%, 45%, and 50%, for the next step of response surface methodology.

[0168] (2) Effect of tea extract addition amount on sensory score of tea beverage

[0169] The effect of different amounts of tea extract added on the sensory scores of birch sap tea beverages, such as Figure 9 As shown in the results, tea extract, as the main component of birch sap tea beverages, can affect their taste and sensory scores. With increasing tea extract content, the sensory scores of the birch sap tea beverage initially increased and then decreased, reaching their optimal taste and highest sensory score at a 40% extraction level. An appropriate proportion of tea extract allows the beverage to achieve an optimal balance in flavor, taste, color, and aroma, resulting in a higher sensory score. Excessive tea extract leads to flavor imbalance, bitterness, and a monotonous aroma, while insufficient tea extract results in a weak flavor, poor color, and insufficient aroma. Therefore, controlling the amount of tea extract added is a key factor in optimizing the sensory quality of birch sap tea beverages. In conclusion, three levels of tea extract addition—35%, 40%, and 45%—were selected for the next step of the response surface methodology experiment.

[0170] (3) The effect of added white sugar on the sensory score of tea beverages

[0171] The effect of different amounts of added white sugar on the sensory scores of birch sap tea beverages, such as Figure 10 As shown in the results, the amount of added white sugar has a significant impact on the sensory score of birch sap tea beverage. With increasing white sugar content, the sensory score initially rises and then falls, reaching its highest value at 3%. Appropriate amounts of white sugar can enhance the flavor, texture, color, and aroma of the beverage, achieving an optimal balance. Excessive white sugar leads to excessive sweetness, a sticky texture, and an unbalanced aroma, while insufficient white sugar results in weak flavor, a thin texture, and a lack of aroma. Therefore, controlling the amount of white sugar added is a key factor in optimizing the sensory quality of birch sap tea beverage. Therefore, response surface methodology was conducted with white sugar addition levels of 2%, 3%, and 4%.

[0172] (4) Effect of citric acid addition on sensory scores of tea beverages

[0173] The effect of different citric acid addition amounts on the sensory scores of birch sap tea beverages, such as Figure 11 As shown in the figure, the addition of 0.2% citric acid provides the beverage with moderate acidity, making it refreshing and non-irritating. This acidity harmonizes with the sweetness of birch sap, the richness of tea, and the sweetness of white sugar, creating a balanced taste experience. When the citric acid addition exceeds 0.2%, the sensory score of the tea beverage shows a downward trend. This is because as the amount of citric acid increases, the acidity of the birch sap tea beverage also increases, causing an imbalance in the sweet and sour ratio and a rough texture. Therefore, three levels of citric acid addition—0.15%, 0.2%, and 0.25%—were selected for the next step of the response surface methodology experiment.

[0174] Example 3 Response Surface Experiment

[0175] Based on the results of previous single-factor experiments, four factors that significantly affected the response variable were selected as independent variables: birch sap addition of 40%, 45%, and 50% (designated as A), tea extract addition of 35%, 40%, and 45% (designated as B), white sugar addition of 2%, 3%, and 4% (designated as C), and citric acid addition of 0.15%, 0.2%, and 0.25% (designated as D). Each factor was assigned three levels, labeled -1, 0, and 1, with sensory scores as the response values ​​(see Table 8). A Box-Behnken design was conducted using the response surface methodology Design-Exper 13, and the results are shown in Tables 9 and 10.

[0176] Table 8. Factors and Levels in Response Surface Experiment

[0177]

[0178] Table 9 Response Surface Experimental Design and Results

[0179]

[0180]

[0181] Table 10. Analysis of Variance and Significance

[0182]

[0183] Note: p<0.01 indicates extremely significant difference (**), p<0.05 indicates significant difference (*).

[0184] A regression model was established using Design Expert 13.0 software, and the experimental data were fitted with a quadratic multivariate serial regression model. Analysis of the sensory evaluation results of the birch sap tea beverage yielded the following regression mathematical model for the amounts of birch sap (A), tea extract (B), white sugar (C), and citric acid (D): Y = 95.2 - 0.375A + 0.6958B + 0.9792C + 0.825D + 0.15AB - 0.025AC + 0.85AD - 0.0875BC + 0.2BD + 0.075CD - 3.1646A 2 -6.1958B 2 -2.4958C 2 -2.8896D 2 .

[0185] The significance test results of the regression model coefficients show that: in the linear term, B, C, and D have a significant impact on the response value; in the quadratic term, A... 2 B 2 C2 D 2 The impact on the response value is extremely significant. Based on the P-values ​​of each factor in this model, A (0.2404), B (0.0391), C (0.0064), and D (0.0173), it can be seen that the order of importance of factors affecting the sensory score of birch sap tea beverage is C>D>B>A, and the amount of added white sugar>citric acid>tea extract>birch sap>beef.

[0186] The response surfaces and contour plots for each factor are shown below. Figure 12-17 .

[0187] from Figure 12 The response surface plots show that the amounts of both birch sap and tea extract significantly affect the sensory scores of the birch sap tea beverage. With increasing birch sap content, the sensory scores initially rise steadily, reach a peak, and then gradually decline. Similarly, changes in the amount of tea extract show a similar fluctuating trend. The contour lines in the figure are elliptical, indicating a clear interaction between the two. At lower birch sap levels, increasing the amount of tea extract significantly improves the sensory scores.

[0188] Figure 13 The response surface plot is slightly tilted. Observing the contour lines, their shape is nearly circular. This indicates an interaction between the amounts of birch sap and white sugar added, but not with... Figure 12 In comparison, this interaction is weaker. Under the experimental conditions corresponding to this figure, the changes in the amount of birch sap and white sugar added do not have a significant combined effect on the sensory score. Within a certain range, changing either factor alone results in a relatively gradual change in the sensory score, without drastic fluctuations due to the interaction between the two.

[0189] from Figure 14 The response surface methodology (RSM) shows that the addition of both birch sap and citric acid significantly affects the sensory scores of the birch sap tea beverage, indicating an interaction between the two. Further observation reveals that the steepness of the RSM corresponding to the addition of citric acid is significantly higher than that corresponding to the addition of birch sap. This suggests that the effect of citric acid on sensory scores is far greater than that of birch sap, a conclusion highly consistent with the ANOVA results in the table. The near-elliptical shape of the contour lines suggests a certain degree of interaction between the two factors. When the citric acid addition is low, increasing the birch sap addition improves the sensory scores to some extent; however, when the citric acid addition is too high, the effect of changes in birch sap addition on sensory scores is limited, and vice versa. A deeper analysis of the RSM reveals a clear peak, further confirming the pairwise interactions between the influencing factors.

[0190] Figure 15 The displayed response surface plot exhibits a distinctly steep trend, indicating that the amounts of tea extract and white sugar added significantly affect the sensory scores of the birch sap tea beverage. The contour lines are typically elliptical, clearly demonstrating a significant interaction between the two. In actual production, small changes in the amount of white sugar added can significantly affect sensory scores when adjusting the amount of tea extract, and vice versa. For example, when the amount of tea extract added is low, a moderate increase in white sugar can significantly improve the sensory score; however, when the amount of tea extract added is too high, even increasing the amount of white sugar may not improve the sensory score, and may even cause it to decrease.

[0191] Figure 16 The steep contour lines indicate that the amounts of both tea extract and citric acid significantly affect the sensory scores of the birch sap tea beverage. The elliptical shape of the contour plots further confirms the significant interaction between the two factors. The experiment revealed that the amount of citric acid added was particularly sensitive to changes in the amount of tea extract. A small amount of tea extract significantly improved the sensory score by increasing the amount of citric acid; however, excessive tea extract and further increases in citric acid could lead to a decrease in the sensory score.

[0192] Figure 17 The response surface plot shows that the amount of added white sugar has a greater impact on the sensory score of birch sap tea beverage than the amount of added citric acid, which is consistent with the results of the analysis of variance in the table. The contour lines in the plot are elliptical, indicating a certain interaction between the two.

[0193] In summary, the response surface methodology results indicate that the optimal formulation for birch sap tea beverage is: 3.197% white sugar, 0.207% citric acid, 40.284% tea extract, and 44.802% birch sap. Under this formulation, the predicted sensory score is 95.382. To verify the model's reliability, the parameters were rounded down to: 3.2% white sugar, 0.21% citric acid, 40.3% tea extract, and 44.8% birch sap. Three repeated experiments were conducted, yielding an average sensory score of 94.89, differing from the predicted value by only 0.492. This demonstrates that the model accurately predicts the optimal ratio.

[0194] Experiment 4: Quality Testing of Tea Beverages

[0195] Coliform bacteria were determined according to the plate count method of Escherichia coli in Method II of GB 4789.3-2016 "National Food Safety Standard for Microbiological Examination of Food"; total bacterial count was determined according to GB 4789.2-2022 "National Food Safety Standard for Microbiological Examination of Food"; tea polyphenol content was determined according to GB / T 21733-2008 "Tea Beverages"; caffeine content was determined according to GB / T5009.139-2014.

[0196] The quality of the birch sap tea beverage prepared in Example 1 was tested. The results showed that all microbiological indicators of the product met the national food safety standards. The specific results are as follows: total bacterial count 45 CFU / mL <100, coliform bacteria not detected, tea polyphenol content 690mg / kg ≥500mg / kg, which meets GB / T 21733-2008 "Tea Beverages", and caffeine content 118mg / kg ≥60mg / kg, which meets GB / T 5009.139-2014.

[0197] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A birch sap tea beverage, characterized in that, 100mL contains the following components: 40-50mL birch sap, 35-45mL tea extract, 2-4g sugar, 0.15-0.25g citric acid, and the remainder water.

2. The birch sap tea beverage as described in claim 1, characterized in that, 100 mL contains the following components: 42-48 mL of birch sap, 38-42 mL of tea extract, 2.5-3.5 g of sugar, 0.18-0.22 g of citric acid, and the remainder water.

3. The birch sap tea beverage as described in claim 1 or 2, characterized in that, The preparation of the tea extract includes the following steps: mixing tea leaves with water, soaking, filtering, and collecting the filtrate.

4. The birch sap tea beverage as described in claim 3, characterized in that, The mass-to-volume ratio of tea leaves to water is 1g:40-60mL.

5. The birch sap tea beverage as described in claim 3, characterized in that, The soaking temperature is 70-90℃ and the soaking time is 15-20 minutes.

6. The birch sap tea beverage as described in claim 3 or 4, characterized in that, The tea mentioned includes green tea, black tea, or oolong tea.

7. The method for preparing the birch sap tea beverage according to any one of claims 1-6, characterized in that, The process includes the following steps: mixing birch sap, tea extract, sugar, and citric acid, adjusting the volume with water, homogenizing, sterilizing, and packaging.

8. The preparation method according to claim 7, characterized in that, The homogenization process is carried out at a temperature of 50-70℃, a pressure of 30-40MPa, and a time of 5-15min.

9. The preparation method according to claim 7, characterized in that, The sterilization temperature is 62-65℃ and the time is 25-35 minutes.

10. The use of the birch sap tea beverage as described in any one of claims 1-6 or the birch sap tea beverage obtained by the preparation method as described in any one of claims 7-9 in the preparation of functional beverage products.

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