Streptococcus thermophilus and lactobacillus fermented flaxseed and peanut meal plant milk as well as preparation method and application thereof

Abstract

The invention discloses streptococcus thermophilus and lactobacillus fermented flaxseed and peanut meal plant milk as well as a preparation method and application thereof. Belongs to the technical field of plant milk fermentation. The optimized combination of streptococcus thermophilus and lactobacillus can stably grow and complete fermentation by taking the flaxseed and peanut meal plant base which does not contain cow milk components as a substrate, so that the problems of slow growth, poor acidity generation, insufficient metabolism and the like of a traditional lactobacillus strain in a plant base environment are solved. By optimizing the fermentation process and accurately controlling the fermentation temperature, time and strain inoculum size, the nutritional quality and flavor of the flaxseed and peanut meal fermented plant milk are improved, the problem of unstable texture caused by essential difference of plant proteins is solved, and the overall sensory acceptance of the plant-based fermented milk is improved.

Description

Technical Field

[0001] This invention relates to the field of plant milk fermentation technology, and more specifically to the fermentation of flaxseed and peanut meal plant milk by Streptococcus thermophilus and Lactobacillus, as well as its preparation method and application. Background Technology

[0002] Plant-based dairy products are made from legumes, nuts, grains, and other raw materials, processed into foods that resemble animal dairy products in appearance, taste, and application. Driven by health and sustainable consumption trends, this type of product has become an important development direction for the dairy industry, and both domestic and foreign companies have entered the market.

[0003] Existing plant-based fermented dairy products mostly use soybeans, oats, coconuts, almonds, peas, etc. as raw materials, employing lactic acid bacteria fermentation and supplemented with food colloids to improve texture. However, they have the following problems: First, plant proteins have poor solubility and low emulsification stability, easily causing precipitation, stratification, and rheological fluctuations, resulting in a rough taste; second, there is a limited number of dedicated fermentation strains, and traditional lactic acid bacteria have insufficient acid production and metabolic capacity in plant substrates, resulting in low fermentation efficiency and limited flavor improvement; third, the products generally have undesirable flavors such as a raw, grassy taste, which limits quality improvement and industrial application.

[0004] Peanut meal has a high crude protein content, approaching 48%, with arginine content as high as 5.2%, making it highly nutritious. Flaxseed is rich in omega-3 high-unsaturated fatty acids and tryptophan. Unsaturated fatty acids are essential nutrients that animals cannot synthesize themselves, while tryptophan helps balance amino acids and improve protein bioavailability. Previous laboratory studies have developed flaxseed plant-based milk, flaxseed fermented milk, and flaxseed-peanut meal plant-based milk. While the combination of flaxseed and peanut meal can improve protein levels to some extent and provide a more comprehensive amino acid profile and essential fatty acids, using peanut meal and flaxseed as plant-based dairy ingredients results in undesirable sensory characteristics such as a raw, grassy taste, affecting the consumer experience. Currently, there is still a lack of specific fermentation strains that can effectively degrade off-flavor substances or produce typical milky flavors (such as lactic acid and dimethyl ethyl ketone) in this type of plant-based system.

[0005] The patent application number 202111294217.4, entitled "A Direct-Inoculation Fermentation Agent for Reducing the Beany Flavor and Its Preparation Method and Application," mentions *Streptococcus thermophilus* ST81-08 and *Lactobacillus bulgaricus* LB42. However, the core objective of this patent is to effectively reduce the content of typical beany flavor substances such as hexanal and pentanal in soybean raw materials through the synergistic effect of *Streptococcus thermophilus* ST81 and *Lactobacillus bulgaricus* LB42, thereby improving the sensory quality of soy milk and making the beany aroma more prominent. It offers no inspiration for solving the aforementioned issues of stability and raw, grassy taste.

[0006] Therefore, how to develop specific strain combinations and fermentation methods for flaxseed and peanut meal plant-based fermented milk, effectively regulate the texture of flaxseed and peanut meal plant-based fermented milk, improve the storage stability of fermented milk, effectively degrade off-flavor substances to enhance the milky aroma, and improve the nutritional value of plant-based fermented milk are technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a plant-based milk fermented from flaxseed and peanut meal using *Streptococcus thermophilus* and *Lactobacillus*, its preparation method, and its application. It is the first discovery of the specificity of the combination of *Streptococcus thermophilus* and *Lactobacillus* in plant-based fermented milk. This strain combination can effectively adapt to flaxseed and peanut meal plant-based fermentation materials, exhibiting high acid production and good growth activity. It can effectively utilize the nutrients in flaxseed and peanut meal to complete the fermentation of plant-based milk, reducing the unpleasant raw, grassy flavor of flaxseed and peanut meal, improving the texture and stability of the product, promoting the formation of flavor compounds, and enhancing the nutritional value of the product. Simultaneously, the mixed fermentation significantly enhances the fruity, milky, and floral aroma characteristics of the product, resulting in a more harmonious and pleasant overall flavor quality.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution: The primary objective of this application is to provide the application of Streptococcus thermophilus and Lactobacillus synergistically in the fermentation preparation of flaxseed and peanut meal fermented plant milk.

[0009] As a preferred technical solution, the thermophilic streptococcus is thermophilic streptococcus ST81-08; the lactobacillus is Lactobacillus bulgaricus LB42; the two are compounded in a mass ratio of 1:3 to 3:1.

[0010] As a more preferred technical solution, the thermophilic streptococcus ST81-08 and Lactobacillus bulgaricus LB42 are compounded in a 1:1 mass ratio.

[0011] Another object of this application is to provide: a microbial agent for fermenting flaxseed and peanut meal into fermented plant milk, comprising Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42; the two are compounded in a mass ratio of 1:3 to 3:1.

[0012] As a preferred technical solution, the microbial agent for fermenting flaxseed and peanut meal into fermented plant milk includes Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42; the two are compounded in a 1:1 mass ratio.

[0013] Another object of this application is to provide a method for preparing a fermented plant milk from flaxseed and peanut meal, comprising the following steps: (1) Preparation of plant milk: Mix flaxseed and peanut meal, soak, grind and filter to remove residue, then add sugar and tocopherol, homogenize and sterilize to prepare plant milk; (2) Post-ripening treatment: Add a fermentation agent of Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42 in a ratio of 1:3-3:1 to the plant milk obtained in step (1), ferment at 45℃ for 4-8h, and refrigerate at 4℃ for 8-12h to prepare flaxseed peanut meal fermented plant milk.

[0014] As a preferred technical solution, the weight ratio of flaxseed to peanut meal in step (1) is 2.5-1.5:1; the soaking time is 1-3 hours. As a preferred technical solution, the grinding time is 8-12 minutes; the filtration is performed using 150-200 mesh gauze. As a preferred technical solution, the amount of sugar added is 3-4%; the amount of tocopherol added is 0.02-0.04%; the homogenization time is 3-5 minutes; the sterilization is performed by pasteurization at a temperature of 85-90°C for 10 minutes.

[0015] As a preferred technical solution, the amount of fermentation agent added in step (2) is 8-10 mg / 100 g.

[0016] As a more preferred technical solution, the amount of fermentation agent added in step (2) is 10 mg / 100 g; In step (2), the thermophilic streptococcus ST81-08 and Lactobacillus bulgaricus LB42 were mixed in a 1:1 ratio; fermented at 45°C for 6 hours, and then refrigerated at 4°C for 12 hours.

[0017] Another object of this application is to provide: flaxseed peanut meal fermented plant milk prepared by any of the above methods.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides the application of thermophilic streptococci and lactobacilli in the fermentation preparation of flaxseed and peanut meal fermented plant milk. The specificity of this combination in flaxseed and peanut meal plant-based fermented milk was discovered for the first time. The above-mentioned strain combination can grow stably and complete fermentation in a plant-based system (flaxseed and peanut meal) that is completely free of milk components, which solves the problems of slow growth, poor acidity generation and insufficient metabolism of traditional lactic acid bacteria strains in plant-based environment.

[0019] (2) In addition, by optimizing the fermentation process and precisely controlling the fermentation temperature, time and inoculation amount, the nutritional quality and sensory evaluation of flaxseed-peanut meal plant fermented milk were further improved, the generation of flavor substances (such as lactic acid, acetaldehyde, etc.) was promoted, and the generation of undesirable flavors was reduced. The problem of textural instability caused by the essential differences in plant proteins was solved, and the overall sensory acceptance of plant-based fermented milk was improved. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 The study investigated the changes in pH, titratable acidity, and viable cell count during the fermentation of flaxseed-peanut meal into plant milk by different thermophilic streptococci.

[0022] Figure 2 The results indicate the changes in pH, titratable acidity, and viable cell count during the fermentation of flaxseed-peanut meal into plant milk using different mixed cultures.

[0023] Figure 3 The study investigated the changes in pH and acidity during the fermentation of flaxseed-peanut meal into plant milk using different ratios of Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42.

[0024] Figure 4 The study investigated the changes in pH and acidity during the fermentation of flaxseed-peanut meal into plant milk by different amounts of Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42.

[0025] Figure 5 The changes in pH and acidity during the fermentation of flaxseed-peanut meal into plant milk by Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42 at different fermentation times were studied.

[0026] Figure 6 The changes in pH and acidity during the fermentation of flaxseed-peanut meal into plant milk by Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42 at different fermentation temperatures were studied.

[0027] Figure 7 The textural characteristics of flaxseed-peanut meal fermented plant milk prepared under optimal fermentation process.

[0028] Figure 8The storage stability of flaxseed-peanut meal fermented plant milk prepared under optimal fermentation process; Note: CK: Unfermented flaxseed-peanut meal fermented milk; LB: Flaxseed-peanut meal fermented plant milk fermented by Lactobacillus bulgaricus LB42; ST: Flaxseed-peanut meal fermented plant milk fermented by Streptococcus thermophilus ST81-08; ST+LB: Flaxseed-peanut meal fermented plant milk fermented by a 1:1 mixture of Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42.

[0029] Figure 9 For: Heatmap analysis of the content of major volatile flavor compounds in flaxseed-peanut meal fermented plant milk. Note: CK: Unfermented flaxseed-peanut meal fermented plant milk; LB: Flaxseed-peanut meal fermented plant milk fermented by Lactobacillus bulgaricus LB42; ST: Flaxseed-peanut meal fermented plant milk fermented by Streptococcus thermophilus ST81-08; ST+LB: Flaxseed-peanut meal fermented plant milk fermented by a 1:1 mixture of Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The strains used in the embodiments of this application are from the following sources: Streptococcus thermophilus ST81-08, Streptococcus thermophilus ST81-67, Streptococcus thermophilus ST81-72, Streptococcus thermophilus ST81-34, Lactobacillus plantarum LP90, Lactobacillus helveticus LH76, Lactobacillus rhamnosus LRa05, Lactobacillus acidophilus La85, and Lactobacillus casei LC89 were all purchased from Microcon Probiotics (Suzhou) Co., Ltd. Taking Streptococcus thermophilus ST81-08 as an example, Streptococcus thermophilus is the strain name, and ST81-08 is the product model.

[0032] Lactobacillus bulgaricus LB42, a strain previously disclosed in other existing technologies prior to the application date, is deposited at the China General Microbiological Culture Collection Center and is classified as Lactobacillus deutschlandia subsp. bulgaricus. Lactobacillus delbrueclii subsp. bulgaricus Its accession number is CGMCCNo.15751, the accession date is May 11, 2018, and the accession address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Example 1 Developing fermentation strains suitable for flaxseed-peanut meal fermentation of plant milk (1) Screening of thermophilic streptococci After fermentation, plant-based milk generally suffers from undesirable flavors such as a rough texture, sourness, and a raw, grassy taste. Based on previous research, we screened four single bacterial strains that can reduce the sour taste, accelerate acid production, and produce a delicate and silky texture after fermenting plant-based raw materials. These strains include *Streptococcus thermophilus* ST81-08, *Streptococcus thermophilus* ST81-67, *Streptococcus thermophilus* ST81-72, and *Streptococcus thermophilus* ST81-34. Each of these strains was used as a probiotic to ferment flaxseed-peanut meal individually, as follows: First, the flaxseed undergoes microwave pretreatment: the flaxseed is moistened to 13%, left to stand at 4℃ for 6-12 hours, and then microwaved to remove cyanogenic glycosides from the raw flaxseed. The peanut meal is then crushed using a pulverizer and passed through a 100-200 mesh sieve for later use.

[0034] The flaxseed fermented milk was prepared according to the previously determined laboratory method: flaxseed and peanut meal were mixed and soaked in purified water at a ratio of 1:8 for 1.5-2 hours. After soaking, the solid and liquid were poured together into a colloid mill and ground for 8-12 minutes. After grinding, the mixture was filtered through 150-200 mesh gauze to remove residue. 3-4% white sugar and 0.02-0.04% tocopherol were added. The mixture was homogenized for 3-5 minutes. Pasteurization was performed at 85℃-90℃ for 10 minutes to obtain flaxseed-peanut meal fermented plant milk. The pasteurized plant milk was then placed in a clean bench with the above-mentioned thermophilic streptococci added at a ratio of 6 mg / 100 g. Fermentation was carried out at 42℃ for 6 hours, followed by refrigeration at 4℃ for 12 hours for final maturation.

[0035] The pH, titratable acidity, and viable cell count of the fermentation system were measured at 2, 4, 6, and 8 hours of fermentation. Figure 1 Simultaneously, after fermentation, the sensory evaluation of the flaxseed-peanut meal fermented plant milk was determined (sensory evaluation was conducted according to internal laboratory standards, with ≥10-12 trained evaluators. Terminology and reference sample training were conducted for acidity, sweetness, peanut / nut aroma, beany flavor, oiliness, consistency, viscosity, crispness, smoothness, graininess, and aftertaste. Appearance, odor, taste, and overall acceptability were also evaluated). The experimental results are shown in Table 1.

[0036] Table 1. Sensory evaluation of flaxseed-peanut meal fermented plant milk by different thermophilic streptococci.

[0037] Results Analysis: From Table 1 and... Figure 1Physicochemical analysis revealed that among the four thermophilic streptococci, the flaxseed-peanut meal fermented plant milk obtained by fermentation with Streptococcus thermophilus ST81-08 had a slightly sour, refreshing, delicate, and good taste. Furthermore, the pH decreased rapidly during fermentation, indicating rapid acid production. The high titratable acidity indicated that the faster the fermentation speed, the higher the yield of organic acids. The high viable cell content indicated a significant growth advantage and rapid strain growth in flaxseed-peanut meal fermentation. Therefore, Streptococcus thermophilus ST81-08 was selected as the suitable strain for flaxseed-peanut meal fermented plant milk, and subsequent experiments were conducted using this strain.

[0038] (2) Screening of compound strains A: Sensory evaluation and fermentation characteristics of flaxseed-peanut meal fermented plant milk Significant differences exist in the growth and acid-producing capacities of different lactic acid bacteria. Mixed lactic acid bacteria fermentation has the potential for synergistic growth. Therefore, *Streptococcus thermophilus* ST81-08 was mixed with *Lactobacillus plantarum* LP90, *Lactobacillus helveticus* LH76, *Lactobacillus bulgaricus* LB42, *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La85, or *Lactobacillus casei* LC89 at a 1:1 mass ratio to explore the advantages of mixed-culture fermentation of flaxseed and peanut meal. The specific process is as follows: First, the flaxseed undergoes microwave pretreatment: the flaxseed is moistened to 13%, left to stand at 4℃ for 6-12 hours, and then microwaved to remove cyanogenic glycosides from the raw flaxseed. The peanut meal is then crushed using a pulverizer and passed through a 100-200 mesh sieve for later use.

[0039] The flaxseed fermented milk was prepared according to the previously determined laboratory method: flaxseed and peanut meal were mixed and soaked in purified water at a ratio of 1:8 for 1.5-2 hours. After soaking, the solid and liquid were poured together into a colloid mill and ground for 8-12 minutes. After grinding, the mixture was filtered through 150-200 mesh gauze to remove residue. 3-4% white sugar and 0.02-0.04% tocopherol were added. The mixture was homogenized for 3-5 minutes. Pasteurization was performed at 85℃-90℃ for 10 minutes to obtain flaxseed-peanut meal fermented plant milk. The pasteurized plant milk was then placed in a clean bench with the above-mentioned thermophilic streptococci added at a ratio of 6 mg / 100 g, and fermented at 42℃ for 6 hours. Afterward, it was refrigerated at 4℃ for 12 hours for final maturation.

[0040] Based on the fermentation characteristics of the strains (pH, acidity, viable cell count), a suitable mixed microbial combination for flaxseed-peanut meal fermentation was screened, and the experimental results are as follows: Figure 2 As shown in Table 2.

[0041] Table 2 Sensory evaluation of flaxseed-peanut meal fermented plant milk with different mixed cultures

[0042] Note: CK: Unfermented flaxseed-peanut meal plant milk; ST+LP90: Mixed fermentation of Lactobacillus plantarum LP90 and Streptococcus thermophilus ST81-08; ST+LH76: Mixed fermentation of Lactobacillus helveticus LH76 and Streptococcus thermophilus ST81-08; ST+LB42: Mixed fermentation of Lactobacillus bulgaricus LB42 and Streptococcus thermophilus ST81-08; ST+LRa-05: Mixed fermentation of Lactobacillus rhamnosus LRa05 and Streptococcus thermophilus ST81-08; ST+LA85: Mixed fermentation of Lactobacillus acidophilus La85 and Streptococcus thermophilus ST81-08; ST+LC89: Mixed fermentation of Lactobacillus casei LC89 and Streptococcus thermophilus ST81-08.

[0043] Results analysis: The study found that all seven mixed strains could decrease the pH of flaxseed-peanut meal after fermentation, and the pH value was between 4.49 and 4.24 6 h after fermentation, indicating that all seven mixed strains could grow well in flaxseed-peanut meal.

[0044] The titratable acidity results were consistent with the pH results; the faster the fermentation rate, the higher the yield of organic acids. All seven mixed bacterial strains increased the titratable acidity after fermenting flaxseed-peanut meal. After 6 hours of fermentation, the titratable acidity increased from 17.5 to 33.5–36°T.

[0045] The seven mixed bacterial strains showed rapid viable cell growth rates from 0 to 4 hours. Among them, *Streptococcus thermophilus* ST81-08 and *Lactobacillus bulgaricus* LB42 exhibited significant growth advantages in flaxseed-peanut meal fermentation, demonstrating not only rapid lactic acid bacteria growth but also reaching a maximum viable cell count of 2.45 × 10⁻⁶ at the end of fermentation. 8 CFU / mL.

[0046] B: Nutritional index testing of flaxseed-peanut meal fermented plant milk Furthermore, in order to determine the effect of mixed strains on the nutritional composition of flaxseed-peanut meal fermented plant milk, the contents of total phenols, flavonoids, protein and fat in the fermented flaxseed-peanut meal fermented plant milk were measured, and the experimental results are shown in Table 3.

[0047] Table 3 Basic Nutritional Indicators of Fermented Plant Milk from Mixed Microbial Fermentation of Flaxseed and Peanut Meal

[0048] Note: CK: Unfermented flaxseed-peanut meal plant milk; ST+LP90: Mixed fermentation of Lactobacillus plantarum LP90 and Streptococcus thermophilus ST81-08; ST+LC89: Mixed fermentation of Lactobacillus casei LC89 and Streptococcus thermophilus ST81-08; ST+La85: Mixed fermentation of Lactobacillus acidophilus La85 and Streptococcus thermophilus ST81-08; ST+LRa05: Mixed fermentation of Lactobacillus rhamnosus LRa05 and Streptococcus thermophilus ST81-08; ST+LB42: Mixed fermentation of Lactobacillus bulgaricus LB42 and Streptococcus thermophilus ST81-08; ST+LH76: Mixed fermentation of Lactobacillus helveticus LH76 and Streptococcus thermophilus ST81-08; DW: Dry weight.

[0049] Results Analysis: The total phenolic, flavonoid, and protein contents in the fermented flaxseed-peanut meal milk after mixed-culture fermentation were higher than those in the unfermented flaxseed-peanut meal milk group. The mixed fermentation of *Lactobacillus bulgaricus* LB42 and *Streptococcus thermophilus* ST81-08 showed the most significant increases in total phenolic, flavonoid, and protein contents. The fat content of the fermented milk decreased in all seven mixed-culture fermentations, indicating that probiotics can secrete amylase and other lipid oxidases during fermentation, breaking down and metabolizing large lipid molecules into flavor compounds. Based on the fermentation characteristics and nutritional index results, *Streptococcus thermophilus* ST81-08 and *Lactobacillus bulgaricus* LB42 were selected as the most suitable mixed strains for fermenting flaxseed-peanut meal.

[0050] Example 2 Optimize fermentation process (1) Investigation on the optimal strain ratio The optimal bacterial strain combination (Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42) was used for flaxseed-peanut meal plant milk fermentation. The fermentation conditions were: inoculum size 6 mg / 100 g, fermentation time 6 h, fermentation temperature 42℃, and bacterial strain ratios (Streptococcus thermophilus ST81-08:Lactobacillus bulgaricus LB42, mL:mL) of 3:1, 2:1, 1:1, 1:2, and 1:3. The optimal bacterial strain ratio was investigated using total acid, total sugar, total phenols, and total flavonoids as indicators, and a comprehensive evaluation value as the indicator. The experimental results are as follows: Figure 3 As shown in Table 4.

[0051] Table 4. Total phenols, flavonoids, and total sugar content in flaxseed-peanut meal fermented plant milk with different strain ratios

[0052] Note: DW: dry weight.

[0053] Results Analysis: When the ratio of *Streptococcus thermophilus* ST81-08 to *Lactobacillus bulgaricus* LB42 was 1:1, the pH value was lower, and the acidity reached a maximum of 52°T. The titration results for acidity were consistent with the pH results, indicating that the 1:1 ratio yielded the best acid production effect. Furthermore, after fermentation, the flavonoid content was higher in the 1:1 ratio group C compared to the unfermented group. Compared to the unfermented group, the total phenol content of some combinations increased slightly (groups D and G). The total sugar content of group C was significantly lower than that of the unfermented group, indicating that lactose was consumed the most and organic acids accumulated, consistent with the acidity results. Therefore, we selected a 1:1 ratio of *Streptococcus thermophilus* ST81-08 to *Lactobacillus bulgaricus* LB42 as the optimal ratio for subsequent experiments.

[0054] (2) Investigation of the optimal strain addition amount The optimal combination of *Streptococcus thermophilus* ST81-08 and *Lactobacillus bulgaricus* LB42 was used for fermentation of flaxseed and peanut mixed plant milk. The experimental conditions were a 1:1 strain ratio, a fermentation time of 6 h, a fermentation temperature of 42℃, and strain addition amounts of 6 mg / 100 g, 8 mg / 100 g, and 10 mg / 100 g. The optimal strain addition amount was investigated using total acid, total sugar, total phenols, and total flavonoids as indicators, and a comprehensive evaluation value as the indicator. The experimental results are as follows: Figure 4 As shown in Table 5.

[0055] Table 5. Total phenols, flavonoids, and total sugar content of flaxseed-peanut meal fermented plant milk with different amounts of starter culture.

[0056] Note: DW: dry weight.

[0057] Results Analysis: Overall acidity showed relatively small changes, remaining above 50°T. The acidity was higher in the group with an addition of 10 mg / 100 g compared to the other two groups. Increased probiotic inoculation led to increased lactic acid accumulation. With increasing mixed bacteria addition, the flavonoid content in the flaxseed and peanut fermented milk increased, with the highest flavonoid content observed in group C at an addition of 10 mg / 100 g. Total phenol content also increased with increasing addition, with group C showing higher levels. The total sugar content in group C was significantly lower than in group A. At an addition of 10 mg / 100 g, probiotics could more effectively break down sugars into flavor compounds. Sensory evaluation of the three groups showed that group C's flavor was significantly superior to groups A and B. Therefore, an addition of 10 mg / 100 g was selected as the optimal addition for subsequent experiments.

[0058] (3) Investigation of the optimal fermentation time The optimal combination of *Streptococcus thermophilus* ST81-08 and *Lactobacillus bulgaricus* LB42 was used for the fermentation of flaxseed and peanut mixed plant milk. The fermentation conditions were: a strain ratio of 1:1, an inoculum size of 10 mg / 100 g, a fermentation temperature of 42℃, and fermentation times of 4 h, 6 h, 8 h, and 10 h. The optimal fermentation time was investigated using total acid, total sugar, total phenols, and total flavonoids as indicators, and a comprehensive evaluation value as the indicator. The experimental results are as follows: Figure 5 As shown in Table 6.

[0059] Table 6. Total phenols, flavonoids, and total sugar content of flaxseed-peanut meal fermented plant milk at different fermentation times

[0060] Note: DW: dry weight.

[0061] Results Analysis: The acidity of the fermented lactic acid increased continuously with fermentation time. The highest acidity (65°T) was observed after 10 hours of fermentation, while the lowest (47°T) was observed after 4 hours. Acidity remained above 50°T after 6 and 8 hours of fermentation. Sensory evaluation showed that group A had an indistinct sweet and sour flavor, group B had a moderate sweet and sour taste and good mouthfeel, groups C and D were too sour and unpalatable, and group D had a thinner texture and received a lower overall rating. The highest flavonoid and total phenolic contents were observed after 6 hours of fermentation, while the total sugar content was slightly higher than that of group C (fermentation time 8 hours). Based on comprehensive evaluation, a fermentation time of 6 hours was the optimal fermentation time.

[0062] (4) Investigation of the optimal fermentation temperature The optimal combination of *Streptococcus thermophilus* ST81-08 and *Lactobacillus bulgaricus* LB42 was used for the fermentation of flaxseed and peanut mixed plant milk. The strain ratio was 1:1, the inoculum size was 10 mg / 100 g, the fermentation time was 6 h, and the fermentation temperatures were 40℃, 42℃, and 45℃. The optimal fermentation temperature was investigated using total acid, total sugar, total phenols, and total flavonoids as indicators, and a comprehensive evaluation value as the indicator. The experimental results are as follows: Figure 6 As shown in Table 7.

[0063] Table 7. Total phenols, flavonoids, and total sugar content of plant milk fermented from flaxseed and peanut meal at different fermentation temperatures.

[0064] Note: DW: dry weight.

[0065] Results Analysis: The acidity was higher at a fermentation temperature of 40℃, and lowest at 45℃ (51°T). This may be because the fermentation rate was relatively faster at 40℃, resulting in a higher lactic acid content. After fermentation at 45℃, the flavonoid and total phenolic contents were higher than the other two groups, while the total sugar content was lower. Sensory evaluation of the three groups showed that the lactic acid from the 45℃ fermentation was moderately sweet and sour, with a mild flaxseed aroma and a smooth texture. The flaxseed flavor was stronger at 40℃, the taste was more sour, and the texture was more viscous. Overall, a fermentation temperature of 45℃ is the optimal fermentation time.

[0066] Example 3 Changes in texture and nutrient composition of fermentation products under optimal fermentation process Based on the optimal fermentation process determined in Example 2, the optimal fermentation conditions were as follows: the ratio of Streptococcus thermophilus ST81-08 to Lactobacillus bulgaricus LB42 was 1:1, the inoculum amount was 10 mg / 100 g, the fermentation time was 6 h, and the fermentation temperature was 45 °C. The specific process is as follows: First, the flaxseed undergoes microwave pretreatment: the flaxseed is moistened to 13%, left to stand at 4℃ for 6-12 hours, and then microwaved to remove cyanogenic glycosides from the raw flaxseed. The peanut meal is then crushed using a pulverizer and passed through a 100-200 mesh sieve for later use.

[0067] The flaxseed fermented milk was prepared according to the previously determined laboratory method: flaxseed and peanut meal were mixed and soaked in purified water at a ratio of 1:8 for 1-3 hours. After soaking, the solid and liquid were poured together into a colloid mill and ground for 8-12 minutes. After grinding, the mixture was filtered through 150-200 mesh gauze to remove residue. 3-4% white sugar and 0.02-0.04% tocopherol were added. The mixture was homogenized for 3-5 minutes. Pasteurization was performed at 85℃-90℃ for 10 minutes to obtain flaxseed-peanut meal fermented plant milk. The pasteurized flaxseed-peanut meal fermented plant milk was then placed in a clean bench with a compound bacterial strain added at a ratio of 10 mg / 100 g (strain ratio 1:1). Fermentation was carried out at 45℃ for 6 hours, followed by refrigeration at 4℃ for 12 hours for post-ripening.

[0068] The effects of mixed-culture fermentation on the nutritional indicators and textural properties of flaxseed-peanut meal were investigated. The experimental results are as follows: Figure 7 As shown in Table 8.

[0069] Table 8. Basic nutrient content of flaxseed-peanut meal fermented plant milk

[0070] Note: DW: Dry weight; Ck: Unfermented flaxseed-peanut meal plant milk; LB42: Flaxseed-peanut meal fermented plant milk fermented with Lactobacillus bulgaricus mixed with LB42; ST81-08: Flaxseed-peanut meal fermented plant milk fermented with Streptococcus thermophilus ST81-08; ST81-08+LB42: Fermented plant milk fermented with Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus mixed with LB42 in a 1:1 ratio.

[0071] Results analysis: After mixed fermentation of *Lactobacillus bulgaricus* LB42 and *Streptococcus thermophilus* ST81-08, the flavonoid content was 2.41 g / 100 g, which was 8.3% higher than that of unfermented bacteria, 2.58% higher than that of *Streptococcus thermophilus* ST81-08 alone, and 3.28% higher than that of *Lactobacillus bulgaricus* LB42 alone. The total phenolic content was 66.07 mg / g, which was 28.33% higher than that of unfermented bacteria and 1.71% higher than that of *Lactobacillus bulgaricus* LB42 alone. The total solids content was 9.70 g / 100 g. The total solids content (TSI) increased by 15.7% compared to unfermented, increased by 0.62% compared to single-strain fermentation with *Streptococcus thermophilus* ST81-08, and increased by 0.87% compared to single-strain fermentation with *Lactobacillus bulgaricus* LB42. The fat content was 2.15 g / 100g, decreasing by 14% compared to unfermented, decreasing by 4.65% compared to single-strain fermentation with *Streptococcus thermophilus* ST81-08, and decreasing by 2.27% compared to single-strain fermentation with *Lactobacillus bulgaricus* LB42. The protein content was 1.80 g / 100g, compared to 1.79 g / 100g in unfermented, decreasing by 0.16% compared to single-strain fermentation with *Streptococcus thermophilus* ST81-08, and decreasing by 0.01% compared to single-strain fermentation with *Lactobacillus bulgaricus* LB42. The lack of significant change may be due to a balance between the probiotics' protein consumption and metabolism. The fat content decreased significantly after fermentation, indicating that probiotics may secrete amylase and other lipid oxidases during fermentation to break down and metabolize large molecules of sugars and lipids into flavor compounds. After mixed-culture fermentation, the hardness increased by 10.5%, the adhesiveness increased by 43.8%, the stickiness increased by 43.8%, and the chewiness decreased by 11.6%. The fermented milk after mixed-culture fermentation had a smooth and delicate texture, and mixed-culture fermentation significantly improved the textural properties of the mixed plant milk.

[0072] Example 4 Storage stability of fermented products under optimal fermentation process In existing technologies, plant-based fermented milk commonly suffers from problems such as continuously increasing acidity during storage, significant post-acidification, whey separation, and textural instability, leading to a decline in flavor and taste over shelf life. To investigate the storage stability of flaxseed-peanut meal fermented plant milk under the optimal fermentation process, the product was stored at 4°C. The pH value, acidity, and water retention during storage were measured at 1, 7, 14, 21, and 28 days. The experimental results are as follows: Figure 8 As shown.

[0073] Results Analysis: The pH of flaxseed-peanut meal gradually decreased with prolonged storage time, reaching 4.26 after 28 days of refrigeration. This may be due to the continued catalytic reaction by proteases produced by lactic acid bacteria, which converts some lactose into lactic acid, resulting in post-acidification. As storage time continued, the titratable acidity in the fermented milk increased, inhibiting the activity of lactic acid bacteria. During the first 21 days of storage, the titratable acidity of the flaxseed-peanut meal fermented milk increased with time, indicating post-acidification. On day 28, the acidity decreased significantly, possibly due to reduced lactic acid bacteria activity and consequently, reduced acid production. Compared to the unfermented group, the mixed-culture fermented flaxseed-peanut meal showed smaller pH and acidity changes and a lower degree of post-acidification during the first 21 days, maintaining a relatively stable acidity and avoiding flavor deterioration and texture damage caused by excessive acidification, thus improving the stability of the flaxseed-peanut meal fermented plant milk.

[0074] During the 28-day storage period, the water-holding capacity initially increased and then decreased. The unfermented sample showed a lower water-holding capacity, possibly because the metabolic activities of microorganisms such as lactic acid bacteria caused denaturation and cross-linking of proteins in the fermented plant milk, forming a more compact and stable network structure. In the early stages of refrigeration, the low pH value allowed casein to continue polymerizing and forming a stable gel, encapsulating water and increasing water-holding capacity. Later, high acidity disrupted the casein gel structure, leading to a decrease in water-holding capacity and whey separation. The fermented group showed an initial increase in water-holding capacity followed by a decrease. This may be because initially, proteins gradually dissolved and dispersed, increasing their water-binding capacity, but over time, the protein structure gradually loosened, weakening its water-binding ability. During storage, the mixed-culture fermentation group had a higher water-holding capacity compared to the unfermented group, indicating that mixed-culture fermentation can significantly improve the water-holding capacity of flaxseed-peanut meal.

[0075] Example 5 An investigation into changes in volatile flavor compounds and non-volatile differential metabolites in fermentation products under optimal fermentation processes. After initial strain screening, *Streptococcus thermophilus* ST81-08 and *Lactobacillus bulgaricus* LB42 were selected as the optimal combination. Following optimization of fermentation conditions, subsequent investigations explored changes in volatile flavor compounds and non-volatile differential metabolites in single-strain and mixed-strain fermentation. UPLC analysis of volatile flavor compounds in flaxseed-peanut meal revealed that 1-decyl alcohol was present alone in the mixed-strain fermentation, exhibiting rose and orange blossom aromas. 5-Methyl-2-furfural was also present alone in the mixed-strain fermentation group, imparting a spicy, sweet, and caramel-like aroma to the fermented flaxseed-peanut meal. Pyrazines are important volatile compounds in flaxseed-peanut meal milk, formed by the condensation reaction of hexanal and amino acids. The condensation product is then degraded by Strecker to produce amino ketones, which undergo self-condensation and oxidation to form pyrazine compounds. 2,5-Dimethyl-3-ethylpyrazine was the most abundant, imparting a coffee-like flavor to the fermented plant milk. After mixed-culture fermentation of flaxseed and peanut meal fermented plant milk, the content of substances exhibiting fruity, creamy, rose and orange blossom aromas, and light cream fragrances significantly increased, while components with pungent or unpleasant green odors, such as 1-butanol and 3-methyl-(E)-2-heptenal, were not detected. Non-targeted metabolomics analysis of the flaxseed and peanut meal fermented plant milk revealed a significant increase in the content of volatile flavor compounds after mixed-culture fermentation (Table 9). Figure 9 For example, the content of 1-heptanol increased by 137.5% compared with the unfermented group, while the content of single-strain fermentation of Streptococcus thermophilus ST81-08 or Lactobacillus bulgaricus LB42 only increased by 27.8% and 51.8% respectively compared with the unfermented group, indicating that the synergistic effect of mixed bacteria is conducive to the accumulation of 1-heptanol and the coordinated improvement of flavor substances.

[0076] Table 9. Content of major volatile flavor compounds in flaxseed-peanut meal fermented plant milk

[0077] Note: The water threshold refers to the lowest concentration of the flavor substance that can be perceived by humans in pure water (as defined in the "Compilation of Odor Thresholds for Air, Water and Other Media" (2011 edition)); CK represents the unfermented control group; LB represents the treatment group fermented with Lactobacillus bulgaricus LB42; ST represents the treatment group fermented with Streptococcus thermophilus ST81-08; ST+LB represents the mixed fermentation treatment group of Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42.

[0078] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0079] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of thermophilic streptococci and lactobacilli in the synergistic fermentation of flaxseed and peanut meal to prepare fermented plant milk.

2. The application according to claim 1, characterized in that, The thermophilic streptococcus is thermophilic streptococcus ST81-08; the lactobacillus is Lactobacillus bulgaricus LB42; the two are compounded in a mass ratio of 1:3 to 3:

1.

3. The application according to claim 2, characterized in that, The thermophilic streptococcus ST81-08 and Lactobacillus bulgaricus LB42 were compounded in a 1:1 mass ratio.

4. A microbial agent for fermenting flaxseed and peanut meal into plant milk, characterized in that, It includes Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42; the two are compounded in a mass ratio of 1:3 to 3:

1.

5. The microbial agent for fermenting flaxseed and peanut meal into plant milk according to claim 4, characterized in that, It includes Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42; the two are compounded in a 1:1 mass ratio.

6. A method for preparing fermented plant milk from flaxseed and peanut meal, characterized in that, Includes the following steps: (1) Preparation of plant milk: Mix flaxseed and peanut meal, soak, grind and filter to remove residue, then add sugar and tocopherol, homogenize and sterilize to prepare plant milk; (2) Post-ripening treatment: Add a fermentation agent of Streptococcus thermophilus ST81-08 and Lactobacillus bulgaricus LB42 in a ratio of 1:3-3:1 to the plant milk obtained in step (1), ferment at 45℃ for 4-8h, and refrigerate at 4℃ for 8-12h to prepare flaxseed peanut meal fermented plant milk.

7. The method for preparing fermented plant milk from flaxseed and peanut meal according to claim 6, characterized in that, The weight ratio of flaxseed to peanut meal in step (1) is 2.5-1.5:1; the soaking time is 1-3 hours. The grinding time is 8-12 minutes; the filtration is performed using 150-200 mesh gauze. The amount of sugar added is 3-4%; the amount of tocopherol added is 0.02-0.04%; the homogenization time is 3-5 min; the sterilization is performed by pasteurization at a temperature of 85-90℃ for 10 min.

8. The method for preparing flaxseed peanut meal according to claim 6, characterized in that, The amount of fermentation agent added in step (2) is 8-10 mg / 100 g.

9. The method for preparing flaxseed peanut meal according to claim 8, characterized in that, The amount of fermentation agent added in step (2) is 10 mg / 100 g; In step (2), the thermophilic streptococcus ST81-08 and Lactobacillus bulgaricus LB42 were mixed in a 1:1 ratio; fermented at 45°C for 6 hours, and then refrigerated at 4°C for 12 hours.

10. The flaxseed and peanut meal fermented plant milk prepared by any one of the methods described in claims 6-9.

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