Lactic acid bacteria strains capable of converting rutin and their application in the fermentation of locust flower yogurt
By using Enterococcus faecalis and Lactococcus galactiae to convert rutin into quercetin or isoquercetin, the problem of unutilized active ingredients in Sophora japonica flowers was solved, and Sophora japonica yogurt with rich nutrition and significant health benefits was prepared.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-08
- Publication Date
- 2026-04-03
AI Technical Summary
Rutin cannot be directly absorbed by the human intestines. It needs to be degraded into quercetin or isoquercetin by intestinal bacteria before it can exert its antiplatelet activity and cytotoxicity. Current yogurt production processes have failed to effectively utilize the active ingredients in Sophora japonica flowers.
Three lactic acid bacteria strains, Enterococcus faecium Y4-1, Enterococcus faecium Y4-2, and Lactococcus petauri Y5-4, were used to convert rutin into quercetin or isoquercetin, which was then applied to the fermentation process of Sophora japonica yogurt.
The prepared locust flower yogurt has a higher flavonoid content, a sweet and sour taste, good coagulation properties, and higher antioxidant and Fe2+ chelating abilities. Its nutritional and health benefits are significantly better than those of yogurt produced with commercial starter cultures.
Smart Images
Figure CN116790445B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, and more particularly to the field of lactic acid bacteria starter cultures and yogurt processing methods, specifically to three lactic acid bacteria strains capable of converting rutin and their application in the fermentation of locust flower yogurt. Background Technology
[0002] Sophora japonica flowers were among the first flowers recognized in my country as having both medicinal and edible uses. They are extremely rich in nutrients, containing 22% protein and 17 kinds of amino acids, as well as various trace elements such as calcium, phosphorus, magnesium, potassium, iron, manganese, zinc, and copper, and a large number of active ingredients, such as flavonoids like rutin. Therefore, Sophora japonica flowers are often used as a raw material for rutin extraction.
[0003] Rutin can improve microcirculation and reduce capillary fragility, and is used clinically as an adjunct treatment for diseases including diabetes, hypertension, and hyperglycemia. However, rutin cannot be directly absorbed by the human intestine; instead, it is degraded by intestinal bacteria before being absorbed. Reports indicate that rutin metabolites degraded by human intestinal bacteria exhibit greater antiplatelet activity and cytotoxicity than their parent compounds. The rutin metabolites are quercetin or isoquercetin; quercetin is effective in relieving asthma and also provides good relief for phlegm, cough, and other discomforts.
[0004] Therefore, how to utilize Sophora japonica flowers and convert its active ingredient rutin into metabolites that can be absorbed by the intestines is crucial for improving the quality, enriching nutrition, and enhancing the biological functions of food that is both food and medicine.
[0005] With economic development, people's material and spiritual needs are constantly increasing, and their awareness of food safety and health is also growing. Dairy products, as a product with high nutritional value, are increasingly favored by consumers. Yogurt, in particular, has gained significant attention due to its unique flavor, high nutritional value, and health benefits, gradually becoming an indispensable snack in people's daily lives. However, with the increasing demands of consumers, yogurt production also needs to keep pace with the times, continuously updating its production processes to ensure that the produced yogurt balances taste and nutritional health. Summary of the Invention
[0006] One objective of this invention is to propose a lactic acid bacteria strain capable of converting rutin, wherein the strain is one of Enterococcus faecium Y4-1, Enterococcus faecium Y4-2, and Lactococcus petauri Y5-4. Enterococcus faecium Y4-1, Enterococcus faecium Y4-2, and Lactococcus petauri Y5-4 were all deposited on May 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCCNo. 27327, CGMCC No. 27328, and CGMCC No. 27329, respectively. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0007] Among them, *Enterococcus faecium* Y4-1 and *Enterococcus faecium* Y4-2 can convert rutin into isoquercitrin. *Lactococcus petauri* Y5-4 can convert rutin into quercetin.
[0008] The second objective of this invention is to propose the application of the above-mentioned lactic acid bacteria strains that can convert rutin in the fermentation of locust flower yogurt.
[0009] Preferably, the lactic acid bacteria strain capable of converting rutin is *Lactococcus petauri* Y5-4.
[0010] The third objective of this invention is to provide a locust flower yogurt, prepared using one of the above-mentioned lactic acid bacteria strains capable of converting rutin and the following steps:
[0011] S1. Wash and remove impurities from the locust flowers, add water and boil, then simmer for 20-30 minutes to extract the juice. Filter to obtain the juice.
[0012] S2. Add the extract and sugar to milk and homogenize to obtain acacia milk; wherein the volume of the extract is 10%-15% of the milk volume and the mass of the sugar is 6%-7% (g / L) of the milk volume.
[0013] S3. After sterilizing the locust flower milk by pasteurization, inoculate it with one of the lactic acid bacteria strains that can convert rutin as described in claim 1 for fermentation. The inoculation amount of the strain is 2%-5% of the locust flower milk. The fermentation temperature is 40℃-42℃ and the fermentation time is 5h-7h. Finally, it is refrigerated for maturation.
[0014] Furthermore, the lactic acid bacteria strain capable of converting rutin is *Lactococcus petauri* Y5-4.
[0015] Furthermore, Lactococcus petauri Y5-4 was inoculated at 3-4% of the sophora milk.
[0016] Compared with the prior art, the technical effects of the present invention are as follows:
[0017] Enterococcus faecium and Lactococcus petauri are both facultative anaerobic lactic acid bacteria. The lactic acid bacteria strains proposed in this invention capable of converting rutin—Enterococcus faecium Y4-1, Enterococcus faecium Y4-2, and Lactococcus petauri Y5-4—belong to the genus Lactococcus, are Gram-positive, and spherical. Specifically, Lactococcus petauri Y5-4 can convert rutin to quercetin, while Enterococcus faecium Y4-1 and Enterococcus faecium Y4-2 can convert rutin to isoquercetin.
[0018] The lactic acid bacteria strains proposed in this invention that can convert rutin, when used as starter cultures (Enterococcus faecium Y4-1, Enterococcus faecium Y4-2, and Lactococcus petauri Y5-4), can metabolize rutin into flavonoids such as quercetin and isoquercetin, thus improving the total flavonoid content of fermented foods. The resulting set-type locust flower yogurt is uniformly pale yellow, with a rich, characteristic locust flower aroma, good coagulation without impurities, no whey separation, a sweet and sour taste, excellent sensory properties, and high DPPH free radical scavenging ability (DPPH free radical scavenging rate of 43.17%), Fe... 2+ Chelating ability (Fe) 2+ The chelation rate (37.57%) and total antioxidant capacity (FRAP value of 0.3821) were superior to those of locust flower yogurt produced with commercial starter culture.
[0019] In summary, the locust flower yogurt prepared by this invention using lactic acid bacteria strains that can convert rutin not only has the rich nutrition, unique flavor, and health benefits of yogurt, but also uses a unique strain that can convert rutin, making the converted rutin easier to absorb and more effective. This allows the functional components in locust flowers to be fully utilized, providing a new type of yogurt with rich nutrition, unique flavor, and health benefits. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0021] Figure 1 Gram staining microscopic image of Enterococcus faecium Y4-1 provided by the present invention.
[0022] Figure 2 Gram staining microscopic image of Enterococcus faecium Y4-2 provided by the present invention.
[0023] Figure 3 Gram-stained microscopic image of Lactococcus petauri Y5-4 provided by this invention.
[0024] Figure 4 Sensory evaluation results of fermented yogurt made from different rutin-convertible strains provided by this invention.
[0025] Figure 5 The results of the DPPH free radical scavenging ability test of fermented yogurt by different rutin-convertible strains provided by the present invention.
[0026] Figure 6 The results of the detection of total antioxidant capacity of fermented yogurt by different rutin-convertible strains provided by this invention.
[0027] Figure 7 The different rutin-convertible strains of yogurt fermented with Fe provided by this invention 2+ Chelation capacity test results. Detailed Implementation
[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely 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.
[0029] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.
[0030] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.
[0031] Example 1: Screening and Identification of Rutin-Transforming Bacterial Strains
[0032] Enterococcus faecium Y4-1, Enterococcus faecium Y4-2, Enterococcus faecium Y4-4, Lactococcus petauri Y5-2, and Lactococcus petauri Y5-4 were cultured in GAM liquid medium at 37°C for 48 h.
[0033] Rutin powder was dissolved in DMSO to a final concentration of 10 mg / mL. Enterococcus faecium Y4-1 (hereinafter referred to as strain Y4-1), Enterococcus faecium Y4-2 (hereinafter referred to as strain Y4-2), Enterococcus faecium Y4-4 (hereinafter referred to as strain Y4-4), Lactococcus petauri Y5-2 (hereinafter referred to as strain Y5-2), and Lactococcus petauri Y5-4 (hereinafter referred to as strain Y5-4) were then inoculated into 0.6 mL of GAM liquid medium containing 4 μL of rutin and anaerobically cultured at 37°C for 72 h.
[0034] Standard solutions of rutin, isoquercitrin, and quercetin were prepared, and the contents of rutin, isoquercitrin, and quercetin in the culture medium were detected using ultra-high performance liquid chromatography-mass spectrometry. The quantitative results are shown in Table 1, where the ratios represent the percentage of each compound compared to the amount of rutin in the initial mixture.
[0035] Table 1. Amounts of rutin, isoquercitrin, and quercetin after incubation with different bacterial strains.
[0036]
[0037] After 72 hours of incubation, all bacteria metabolized over 90% of the rutin, leaving less than 10 mmol of rutin compared to the initial 99.67 mmol. Overall, incubating rutin with these bacteria yielded more isoquercitrin and quercetin. Strain Y5-4 converted more than 10% of the rutin to quercetin, while isoquercitrin production was less than 1%. However, strains Y4-1 and Y4-2 showed relatively better ability to convert rutin to isoquercitrin, with strain Y4-2 being the most suitable for isoquercitrin production.
[0038] Gram staining and preliminary bacterial morphological examination of strains Y4-1, Y4-2, and Y5-4 revealed them to be Gram-positive cocci. Figure 1 After BLAST analysis of the 16S rDNA gene sequence, strains Y4-1 and Y4-2 were identified as Enterococcus faecium, and strain Y5-4 was identified as Lactococcus petauri.
[0039] Example 2: Application of Rutin-convertible Lactic Acid Bacteria in Yogurt
[0040] This experiment used locust flowers, which are rich in rutin, as an ingredient and lactic acid bacteria that can convert rutin as a starter culture to prepare set-type locust flower yogurt. The preparation process is as follows:
[0041] S1. Wash 20-30g of locust flowers, remove impurities, add 200-300ml of water, bring to a boil, keep simmering for 20-30 minutes, filter and collect the juice;
[0042] S2. Add the extract and sugar to milk and homogenize to obtain acacia milk; wherein the volume of the extract is 10%-15% of the milk volume and the mass of the sugar is 6%-7% (g / L) of the milk volume.
[0043] S3. After pasteurizing the locust flower milk, commercial starter cultures (Lactobacillus bulgaricus and Streptococcus thermophilus), strains Y4-1, Y4-2, and Y5-4 were added for fermentation. The commercial starter cultures were inoculated at 2% of the locust flower milk, while strains Y4-1, Y4-2, and Y5-4 were inoculated at 3%-4%. The fermentation temperature was 40℃-42℃, and the fermentation time was 5-7 hours. Finally, the milk was refrigerated for maturation. Meanwhile, locust flower yogurt fermented only with commercial starter cultures and yogurt fermented only with commercial starter cultures but without locust flowers were used as controls.
[0044] The finished product of acacia yogurt was evaluated based on four aspects: color and flavor, whey texture, consistency, and mouthfeel. The sensory evaluation criteria are shown in Table 4, with a maximum score of 100 points.
[0045] Table 2 Sensory Evaluation Scoring Criteria for Yogurt
[0046]
[0047] The results are as follows Figure 4As shown, the sensory scores, from highest to lowest, are: strain Y5-4, strain Y4-2, and strain Y4-1. Yogurt made with strain Y4-2 has a slightly bitter taste; yogurt made with strain Y4-1 has a strong bitter taste and poor texture; yogurt made with strain Y5-4 has no unpleasant taste, good texture, and better whey appearance than the other two types. Sensory evaluation suggests that strain Y5-4 is more suitable as a starter culture.
[0048] Based on sensory evaluation scores, the optimal process parameters for preparing locust flower yogurt using strain Y5-4 as the starter culture were determined through single-factor experiments: fermentation temperature 42℃, inoculum size 4% of locust flower milk, volume of locust flower extract 15% of milk volume, sugar 6% of milk volume, and fermentation time 6 hours. The resulting set-type locust flower yogurt is uniformly pale yellow, has a rich and distinctive aroma, good coagulation with no impurities, no whey separation, and a sweet and sour taste. All sensory aspects of the yogurt are excellent.
[0049] Example 3: Antioxidant activity of locust flower yogurt prepared using lactic acid bacteria capable of converting rutin.
[0050] Using locust flower yogurt fermented with only commercial starter culture as a control, set-type locust flower yogurt was prepared using three rutin-converting bacterial strains, Y4-1, Y4-2, and Y5-4, according to the method in Example 2. The antioxidant activity of the locust flower yogurts was then tested. The results are as follows: Figures 5-7 As shown.
[0051] The results of the DPPH free radical scavenging ability test of fermented yogurt by different rutin-converting strains are as follows: Figure 5 As shown, the DPPH radical scavenging rate of locust flower yogurt fermented with commercial starter culture was 21.43%. Three rutin-converting bacterial strains enhanced the antioxidant activity of the samples to varying degrees, with the effect on improving the antioxidant activity of the yogurt from strongest to weakest being: strain Y5-4 > strain Y4-2 > strain Y4-1. When 1% of strain Y5-4 was added, the DPPH free radical scavenging rate increased by 10.43%. It can be seen that compared to the control group with only commercial starter culture, strain Y5-4 significantly improved the DPPH free radical scavenging ability of the yogurt.
[0052] Results of total antioxidant capacity testing of fermented yogurt from different rutin-converting strains are as follows: Figure 6 As shown, compared with yogurt fermented with only commercial starter culture (FRAP value 0.2821), fermentation with rutin-convertible strains can enhance the total antioxidant activity of yogurt. Different starter cultures have different effects, and their ability to improve antioxidant activity from high to low is: strain Y5-4 > strain Y4-2 > strain Y4-1, and the effect of using strain Y5-4 is much greater than that of strain Y4-1.
[0053] Fe in yogurt fermented by different rutin-converting strains 2+Chelation capacity test results are as follows Figure 6 As shown, compared with the Fe2+ chelation rate (20.84%) of locust flower yogurt made with only commercial starter culture, the yogurt fermented with rutin-convertible strains had a higher Fe2+ chelation capacity, with strain Y5-4 showing the highest Fe2+ chelation rate. 2+ The chelation rate can reach 34.63%.
[0054] The locust flower yogurt fermented according to the optimal process parameters for preparing locust flower yogurt using strain Y5-4 as the starter in Example 2 had a DPPH free radical scavenging rate of 43.17%, a FRAP value of 0.3821, and Fe... 2+ The chelation rate was 37.57%, which is superior to that of locust flower yogurt produced with commercial starter culture.
[0055] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.
Claims
1. A lactic acid bacteria strain capable of converting rutin, characterized in that, The lactic acid bacteria strain is Enterococcus faecalis ( Enterococcus faecium Y4-1, Enterococcus faecalis ( Enterococcus faecium) Y4-1 was deposited on May 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27327.
2. The application of the lactic acid bacteria strain capable of converting rutin as described in claim 1 in the fermentation of locust flower yogurt.
3. A type of locust flower yogurt, characterized in that, It was prepared using the lactic acid bacteria strain capable of converting rutin as described in claim 1 and the following steps: S1. Wash and remove impurities from the locust flowers, add water and boil, then simmer for 20-30 minutes to extract the juice. Filter to obtain the juice. S2. Add the extract and sugar to milk and homogenize to obtain acacia milk; wherein the volume of the extract is 10%-15% of the milk volume, and the mass of the sugar is 6%-7% of the milk volume. S3. After sterilizing the locust flower milk by pasteurization, inoculate it with the lactic acid bacteria strain that can convert rutin as described in claim 1 for fermentation. The inoculation amount of the strain is 2%-5% of the locust flower milk. The fermentation temperature is 40℃-42℃ and the fermentation time is 5 h-7 h. Finally, it is refrigerated for maturation.
4. The locust flower yogurt according to claim 3, characterized in that, The inoculation amount of the bacteria should be 3-4% of that used for Sophora japonica milk.