Preparation method of fermentative lotus root starch by using Witzia ciliata inm3206 and application of fermentative lotus root starch in tapioca pearls

Abstract

The invention relates to a preparation method of fermented lotus root starch of Weizmannia coagulans and an application of the fermented lotus root starch of Weizmannia coagulans in tapioca pearls, the strain is Weizmannia coagulans, the preservation number is GDMCC NO: 67224, the preservation date is November 6, 2025, the preservation unit is Guangdong Microbial Culture Collection Center, and the preservation address is No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. The method has the advantages that the content of amylopectin in the lotus root starch can be effectively increased by the Witzia ciliaris; the lotus root starch prepared from the Witzia ciliaris can improve the freezing resistance and taste characteristics of the pearl tapioca.

Description

Technical Field

[0001] This invention patent relates to the preparation of lotus root powder fermented by Weizmannii inm3206 and its application in tapioca pearls. Background Technology

[0002] Lotus root starch, a traditional Chinese medicine, is a starch processed from the rhizome of the lotus plant (Nelumbo nucifera), a member of the Nymphaeaceae family. Distributed throughout China, it is also known as lotus root starch. It is sweet and salty in taste, and neutral in nature, entering the spleen, liver, and kidney meridians. It has the effects of nourishing blood, stopping bleeding, regulating the middle jiao (digestive system), and stimulating appetite. It is mainly used to treat weakness and blood loss, diarrhea, and poor appetite. Modern research shows that lotus root contains active substances such as polysaccharides and polyphenols, which can enhance antioxidant capacity, lower blood lipids, and alleviate intestinal inflammation. Its antioxidant and anti-inflammatory effects are significantly enhanced after cooking.

[0003] Tapioca pearls, also known as pearl balls, are a type of snack food with a translucent appearance and high nutritional value. They are easy to cook, convenient to transport, and have a long shelf life. They also have a smooth, chewy, and elastic texture, making them a unique and appealing addition to any dessert or beverage. Tapioca pearls are primarily made from tapioca starch, which contains 83% amylopectin and 17% amylose. This ratio gives them high viscosity and good transparency, making them widely used in food processing. However, pure lotus root starch contains approximately 70% amylose and 30% amylopectin, which makes it prone to aging, has poor water solubility, and weak freeze resistance. As a result, tapioca pearls made from lotus root starch tend to form a hard core in cold drinks. Therefore, increasing the amylopectin content of lotus root starch is a crucial way to prevent a hard core in tapioca pearls made from it.

[0004] Currently, the main method for converting amylose into amylopectin relies on starch branching enzymes. Patent CN200780050622.3, titled "Enzyme-Catalyzed Preparation Method of Highly Branched Amylose and Amylopectin Clusters," relates to an enzyme-catalyzed method for preparing highly branched amylose and amylopectin clusters. α-glucan transferase or branching enzymes hydrolyze the connections between segments of amylopectin clusters in starch, thereby producing amylopectin clusters. Simultaneously, the branching enzyme attaches branched side chains to amylose, producing branched amylose. Subsequently, the amylopectin clusters or branched amylose are treated with maltose amylase to cleave long side chains into shorter ones and transfer glucose to the side chains, thus effectively producing highly branched amylopectin clusters, highly branched amylose, or branched oligosaccharides from starch. However, this method does not involve microbial fermentation modification of lotus root starch, and the branching enzymes involved have not received national approval. Patent: A Method for Preparing and Applying Modified Short Amylose - CN201810290482.7. This invention discloses a method for preparing and applying modified short amylose, belonging to the field of nanocomposite materials technology. This invention utilizes chloroacetic acid to modify short amylose, introducing carboxyl groups into the short amylose and increasing its negative potential. This invention uses carboxymethyl short amylose as a carrier to introduce chitosan for insulin encapsulation, which has the characteristics of wide availability, low price, and high encapsulation rate, achieving excellent results in insulin encapsulation. The preparation process of carboxymethyl short amylose in this invention is green and environmentally friendly, with mild reaction conditions, a simple and easy-to-implement process, and is easy to industrialize, possessing the potential for large-scale application. This invention mainly reduces the length of amylose for use as a carrier and does not involve bio-fermentation modification.

[0005] Currently, there are no technologies related to the application of microbial fermentation to convert the amylose in lotus root powder into amylopectin. Summary of the Invention

[0006] The purpose of this invention is to solve existing technical problems by proposing a method for preparing lotus root powder fermented by Weizmannii inm3206 and its application in tapioca pearls.

[0007] To achieve the above objectives, the present invention employs the following technical solution: a strain of *Weizmannia coagulans*, specifically strain *Weizmannia coagulans* inm3206 (… Weizmannia coagulans The accession number is GDMCC NO: 67224, the accession date is November 6, 2025, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, and the depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The preparation of fermented lotus root powder using *Weizmannia coagulans* involves inoculating lotus root powder with *Weizmannia coagulans*, fermenting at 37°C for 48 hours, and using inoculated fermentation bacteria at concentrations of 10... 4-10 8 CFU / mL.

[0009] Application of a type of lotus root powder fermented with Weizmann's coagulation bacteria in tapioca pearls.

[0010] Furthermore, (1) Raw materials: fermented lotus root powder, tapioca flour, hot water, and white sugar; (2) Production process: (a) Kneading the dough: Pour the white sugar into the tapioca flour and fermented lotus root flour and mix. Slowly add hot water while stirring quickly until it forms clumps. After the temperature drops slightly, knead it by hand into a smooth, non-sticky dough. (b) Rolling and cutting: Divide the dough into small portions, roll each portion into a thin strip, and then cut it into small squares; (c) Rounding to prevent sticking: Sprinkle a little dry tapioca flour on the small squares, gently shake the container, and let the small squares roll naturally into round pearls, while avoiding sticking; (d) Boiling and cooling: Add water to a pot and bring it to a boil. Add the pearls and gently stir with a spoon to prevent them from sinking. After the pearls float to the surface, continue to boil for 8-10 minutes, then turn off the heat and let it sit for 5-8 minutes. (e) Finally, remove the pearls, rinse them with ice water, and drain them.

[0011] The beneficial effects of this invention are: 1. The aforementioned *Weizmannii coagulating* can effectively increase the amylopectin content in lotus root starch; 2. The lotus root powder prepared by the aforementioned *Weizmannii coagulating* can improve the freeze resistance and texture of tapioca pearls. 3. It can increase the content of amylopectin and will not have any off-odor after fermentation. It can be mixed with other food ingredients in the later stage, which not only preserves the nutrition and flavor of lotus root powder, but also enhances its antifreeze properties. Attached Figure Description

[0012] Figure 1 This is a process route diagram.

[0013] Figure 2 This is a colony diagram of *Weizmannii inm3206*.

[0014] Figure 3 This image shows the morphology of *Weizmannii inm3206*. Detailed Implementation

[0015] The present invention will be further described below with reference to specific embodiments: Unless otherwise specified, the experimental methods used in the following implementation examples are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified.

[0016] Example 1 combined with appendix Figure 1-3 A strain of *Weizmannii coagulans*, specifically strain inm3206 (… Weizmannia coagulans The accession number is GDMCC NO: 67224, the accession date is November 6, 2025, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, and the depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0017] A strain of *Weizmann's coagulation* exhibits the following colony characteristics: its colonies are round, off-white or slightly yellowish and opaque, approximately 1-2 mm in diameter, with a rough surface. This bacterium is a Gram-positive bacillus with apical spores and no flagella.

[0018] The preparation of fermented lotus root powder using *Weizmannia coagulans* involves inoculating lotus root powder with *Weizmannia coagulans*, fermenting at 37°C for 48 hours, and using inoculated fermentation bacteria at concentrations of 10... 4 -10 8 CFU / mL.

[0019] Application of a type of lotus root powder fermented with Weizmann's coagulation bacteria in tapioca pearls.

[0020] An application of fermented lotus root powder by Weizmann's coagulation bacteria in tapioca pearls, (1) Raw materials: fermented lotus root powder, tapioca flour, hot water, white sugar; (2) Production process: (a) Kneading the dough: Pour the white sugar into the tapioca flour and fermented lotus root flour and mix. Slowly add hot water while stirring quickly until it forms clumps. After the temperature drops slightly, knead it by hand into a smooth, non-sticky dough. (b) Rolling and cutting: Divide the dough into small portions, roll each portion into a thin strip, and then cut it into small squares; (c) Rounding to prevent sticking: Sprinkle a little dry tapioca flour on the small squares, gently shake the container, and let the small squares roll naturally into round pearls, while avoiding sticking; (d) Boiling and cooling: Add water to a pot and bring it to a boil. Add the pearls and gently stir with a spoon to prevent them from sinking. After the pearls float to the surface, continue to boil for 8-10 minutes, then turn off the heat and let it sit for 5-8 minutes. (e) Finally, remove the pearls, rinse them 2-3 times with ice water or cool water, and drain them.

[0021] The probiotic selection method and fermentation preparation agent described in this invention are as follows: Strain screening: Different strains were cultured to the logarithmic growth phase, and then diluted to a concentration of 10. 5 -10 6 Then, the culture medium was inoculated with 1% of the bacteria into a semi-solid culture medium prepared from lotus root powder and cultured at 37°C for 48 hours to terminate fermentation. Amylopectin was extracted and detected according to the instructions of the amylopectin content kit.

[0022] Production Applications: (1) According to the pearl tapioca pearl production process, fermented lotus root powder was prepared into pearl tapioca pearls, which were then placed in milk tea and placed at 4℃ for 1-8 h. The physicochemical and sensory indicators of the pearl tapioca pearls were then tested.

[0023] (2) According to the preservation method of tapioca pearls, fermented lotus root powder was prepared into tapioca pearls, soaked in sugar solution, and placed at -20℃ to -80℃. The physicochemical indicators and sensory indicators of the tapioca pearls were tested.

[0024] LB medium (g / L): 10.0g tryptone, 5.0g yeast extract, and 10.0g sodium chloride were dissolved in 1000 mL of water and sterilized at 121°C for 20 min.

[0025] The screening medium was formulated as follows: lotus root powder to water in a ratio of 1:1.2 (w / w), mixed thoroughly. Sterilized at 105℃ for 20 min.

[0026] 1. Preliminary screening of bacterial strains Nine bacterial strains preserved in the laboratory were inoculated into LB liquid medium at a 1% inoculum concentration and cultured at 37°C for 24 hours. Then, a 1% inoculum was added to selection medium, with an initial colony concentration of 1.0 × 10⁻⁶. 6 cfu / mL, and statically incubated at 37℃ for 48 h.

[0027] Samples were processed according to the instructions of the amylopectin content kit, and the results are shown in Table 1. Table 1 Analysis of fermented lotus root powder

[0028] As shown in Table 1, strains PC1006, PC1007, and PC1009 with high amylopectin content were selected for secondary screening.

[0029] 2. Secondary screening of strains The initially screened strains PC1006, PC1007, and PC1009 were reactivated and cultured at a cell concentration of 10. 6 CFU / mL bacteria were inoculated into sterilized lotus root powder, fermented at 37℃ for 48 hours, and the ratio of amylose to amylopectin was determined. After being made into tapioca pearls, sensory evaluation was conducted.

[0030] Tapioca pearl making process: (1) Ingredients: 100g fermented lotus root starch, 20g tapioca starch, 40-50ml of boiling water or freshly boiled hot water (must be used to fully gelatinize the starch), 10g white sugar (2) Production process: (a) Kneading the dough: Pour the white sugar into the tapioca starch and fermented lotus root starch and mix. Slowly add hot water while stirring quickly with chopsticks until it forms clumps. After the temperature drops slightly, knead it by hand into a smooth, non-sticky dough.

[0031] (b) Rolling and cutting: Divide the dough into 3-4 small portions, roll each portion into a thin strip with a diameter of 0.8 cm, and then cut it into 1 cm squares.

[0032] (c) Rolling to prevent sticking: Sprinkle a little dry tapioca starch on the small cubes, gently shake the container, and let the small cubes roll into a ball shape naturally to prevent them from sticking together.

[0033] (d) Boiling and Cooling: Bring water to a boil in a pot, add the pearls, and gently stir with a spoon to prevent them from sinking. Once the pearls float to the surface, continue boiling for 8-10 minutes, then turn off the heat and let them sit for 5-8 minutes. Finally, remove the pearls, rinse them 2-3 times with ice water or cold water, and drain.

[0034] Table 2 Sensory evaluation of tapioca pearls

[0035] Note: Each indicator has a maximum score of 5 points, and the higher the score, the better the result.

[0036] As shown in Table 2, PC1006 had the lowest amylose content and the highest amylopectin content, resulting in the best sensory evaluation. Therefore, this strain was selected as the target strain for further research. The morphological observation of this target strain on solid LB agar plates is as follows: Figure 2 As shown, the colonies are round, off-white or slightly yellowish, opaque, approximately 1-2 mm in diameter, with a rough surface. This bacterium is a Gram-positive bacillus with apical spores and no flagella. Gram staining followed by microscopic observation yielded the following results: Figure 3 As shown.

[0037] The PC1006 strain obtained through the above screening was identified by 16S rDNA sequencing. Comparative analysis showed that the 16S region sequence of the strain obtained in this invention was similar to that of *Weizmannella coagulans*. Weizmannia coagulans The similarity reached 100.00%, confirming that this strain is *Weizmannia coagulans*, suitable for use in the food fermentation industry. It is named *Weizmannia coagulans* inm3206. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 6, 2025, with accession number CGMCC No. 67224. Therefore, in this application, INM3206 and PC1006 refer to the same strain. The PC1006 strain of this invention has been genetically identified, and its gene sequence is as follows: Example 2: Detection of genetic stability of strains The strain PC1006 from Example 1 was continuously passaged 10 times on LB liquid medium. The samples from the 1st, 5th, and 10th generations were inoculated into the screening medium, and the ratio of amylopectin to amylose was detected using an amylopectin content kit. At the same time, the samples were re-screened according to Example 1 and prepared into tapioca pearls for sensory evaluation to determine their passage stability. The specific data are shown in Table 3.

[0038] Table 3. Results of the passage stability test for *Wietzmannii coagulans* inm3206

[0039] As shown in Table 3, the fermentation performance of strain PC1006 of different generations was normal, and the proportion of amylopectin fluctuated within 10%. After being made into tapioca pearls, there was no significant change in taste and flavor, indicating that the strain has good genetic stability and meets the requirements for production and use.

[0040] Example 3: Preparation of fermented lotus root powder with different concentrations of *Weizmannii coagulans* inm3206 The *Weizmannii coagulating* inm3206 was inoculated into lotus root powder and fermented at 37°C for 48 hours. Pearl pearls were then prepared according to the method in Example 1. The concentration of the inoculated fermentation bacteria was 10... 4 -10 8 At CFU / mL, tapioca pearls were prepared separately, and PC1002 was used as a control strain. The content of amylopectin, amylose and taste in lotus root starch were detected, and the results are shown in Table 4.

[0041] Table 4. Proportion of amylopectin and amylose in fermented lotus root powder

[0042] Table 5 Sensory evaluation results of tapioca pearls made from fermented lotus root starch

[0043] Note: Each assessment item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all scores. The results in Tables 4 and 5 show that the inoculation of *Weizmannii* inm3206 into fermented lotus root starch significantly increases the amylopectin content while decreasing the amylose content. Furthermore, the higher the inoculation amount, the higher the amylopectin content and the lower the amylose content. However, when processing it into tapioca pearls, excessively high or low inoculation concentrations of *Weizmannii* inm3206 affect the sourness, sweetness, and texture of the tapioca pearls. The optimal inoculation concentration is 1.0 × 10⁻⁶. 7 CFU / mL, this concentration is sufficient to produce enough amylopectin and improve the texture of tapioca pearls.

[0044] Example 4: Effect of tapioca pearls on the antifreeze properties of milk tea at different times The *Weizmannii coagulation* inm3206 strain was inoculated into lotus root powder at an initial concentration of 10. 7 CFU / mL, fermented at 37℃ for 48h, and prepared into tapioca pearls according to the method of Example 1. PC1002 was used as the control strain. The tapioca pearls were placed in milk tea and placed at 4℃ for 1.0, 2.0, 4.0 and 8.0 h to measure the hardness, elasticity, crispness and taste of the pearls. The results are shown in Tables 6 and 7.

[0045] Texture analyzer testing conditions: Use a P / 36R probe, take 3 cooked tapioca pearls, place them in the stage to form a triangle, and ensure that the probe is in the middle of the triangle to start the test. Repeat the test 8 times.

[0046] Table 6. Effects of different refrigeration times on tapioca pearls

[0047] Table 7. Sensory evaluation results of tapioca pearls under different refrigeration times

[0048] As shown in Tables 6 and 7, when tapioca pearls made from lotus root starch fermented with Weizmannii inm3206 were added to milk tea and placed at 4°C, the hardness, brittleness, elasticity, and sensory properties of the tapioca pearls changed over time. However, the overall data of the experimental group remained relatively stable, while the tapioca pearls in the control group became significantly harder, brittler, and lost their elasticity. This is because the fermented lotus root starch showed an increase in amylopectin and a decrease in amylose, which altered the structure of the lotus root starch and made the tapioca pearls made from it more resistant to freezing.

[0049] Example 5: Effect of different freezing temperatures on the freeze resistance of tapioca pearls The *Weizmannii coagulation* inm3206 strain was inoculated into lotus root powder at an initial concentration of 10. 7 CFU / mL, fermented at 37℃ for 48 h, and tapioca pearls were prepared according to the method in Example 1. PC1002 was used as a control strain. The tapioca pearls were added to a sugar solution with a Brix value of 25, and the ratio of tapioca pearls to water was 65:35. The tapioca pearls were placed at -20℃, -40℃, and -80℃ respectively, and their hardness, elasticity, crispness, and texture were tested. The results are shown in Tables 8 and 9.

[0050] Texture analyzer testing conditions: Referring to Example 4, before testing, the frozen tapioca pearls were placed in hot water at 80~90℃ and microwaved for 2 minutes. After cooling, the tapioca pearls were taken out for testing.

[0051] Table 8. Effects of different freezing temperatures on tapioca pearls

[0052]

[0053] Table 9 Sensory evaluation results of tapioca pearls at different freezing temperatures As shown in Tables 8 and 9, when tapioca pearls made from lotus root starch fermented with Weizmannii inm3206 were added to milk tea and placed at temperatures ranging from -20℃ to -80℃, the hardness, brittleness, elasticity, and sensory properties of the tapioca pearls changed as the temperature decreased. However, the overall data of the experimental group remained relatively stable, while the tapioca pearls in the control group became significantly harder, brittler, and lost their elasticity. This is because the fermented lotus root starch showed an increase in amylopectin and a decrease in amylose, which altered the structure of the lotus root starch and made the tapioca pearls made from it more resistant to freezing.

[0054] Based on the results of the above embodiments, the lotus root powder fermented with Weizmannii inm3206 provided by the present invention, when applied to the production of tapioca pearls, can significantly improve the freeze resistance of tapioca pearls without changing the existing process, and at the same time improve the taste of lotus root powder tapioca pearls, providing a theoretical basis for the development of tapioca pearls made from different starch raw materials.

[0055] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A strain of *Weizmannii* coagulans, characterized by: This strain is *Weizmannii coagulans* inm3206. Weizmannia coagulans The accession number is GDMCC NO: 67224, the accession date is November 6, 2025, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, and the depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A method for preparing fermented lotus root powder using *Weizmannii coagulans* as described in claim 1, characterized in that: The *Wietzmannii coagulans* bacteria were inoculated into lotus root powder and fermented at 37°C for 48 hours. The concentration of the inoculated fermentation bacteria was 10... 4 -10 8 CFU / mL.

3. The application of lotus root powder fermented with *Weizmannii coagulatingis* as described in claim 2 in tapioca pearls.

4. The application of the *Wietzmannii* coagulant-producing fermented lotus root powder according to claim 3 in tapioca pearls, characterized in that: (1) Ingredients: fermented lotus root powder, tapioca flour, hot water, and white sugar; (2) Production process: (a) Kneading the dough: Pour the white sugar into the tapioca flour and fermented lotus root flour and mix. Slowly add hot water while stirring quickly until it forms clumps. After the temperature drops slightly, knead it by hand into a smooth, non-sticky dough. (b) Rolling and cutting: Divide the dough into small portions, roll each portion into a thin strip, and then cut it into small squares; (c) Rounding to prevent sticking: Sprinkle a little dry tapioca flour on the small squares, gently shake the container, and let the small squares roll naturally into round pearls, while avoiding sticking; (d) Boiling and cooling: Add water to a pot and bring it to a boil. Add the pearls and gently stir with a spoon to prevent them from sinking. After the pearls float to the surface, continue to boil for 8-10 minutes, then turn off the heat and let it sit for 5-8 minutes. (e) Finally, remove the pearls, rinse them with ice water, and drain them.

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