Method for producing fermented food using a novel Rhizopus microsporus strain, and fermented food

Abstract

To provide a method for making an alternative protein source with a high nutritional value.SOLUTION: A method includes the steps of: providing an isolated and purified Rhizopus microsporus strain, with its deposit number being DSM 34400; and inoculating the isolated and purified Rhizopus microsporus strain to a substrate for fermentation, to form a fermented food. The substrate includes a legume, a processing residue thereof, or a combination thereof.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Taiwan Patent Application No. 110149312, filed on December 29, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to an isolated and purified novel Rhizopus microsporus strain, a method for producing a fermented food using the isolated and purified novel Rhizopus microsporus strain, and the fermented food produced thereby. [Background technology]

[0003] Meat has long been the primary source of protein for humans. With economic growth and an increasing world population, the demand for meat products is also increasing. However, the carbon emissions from meat production are extremely high, and the waste from many livestock releases large amounts of greenhouse gases. Specifically, when ruminants (e.g., cows and sheep) chew their cud, they produce large amounts of methane gas in their intestines. The impact of methane on global warming is about 20 times higher than that of carbon dioxide, and its impact on the worsening of the climate crisis is significant.

[0004] In recent years, due to people's pursuit of healthier diets and the increasing attention to animal welfare and environmental friendliness, the challenge of finding alternative protein sources other than meat and diversifying protein sources has increasingly attracted global attention. Alternative protein sources include, for example, plant-derived proteins, insect-derived proteins, bioengineered artificial meat, and proteins produced by bacterial fermentation. However, the development of nutritious alternative protein sources remains one of the research goals in related fields. Summary of the Invention [Problem to be solved by the invention]

[0005] According to an embodiment of the present disclosure, there is provided an isolated and purified novel Rhizopus microsporus strain, which has a deposit number of DSM 34400.

[0006] According to an embodiment of the present disclosure, there is provided a method for producing a fermented food product using a Rhizopus microsporus strain, the method comprising the steps of: providing an isolated and purified Rhizopus microsporus strain having a deposit number of DSM 34400; and inoculating and fermenting the isolated and purified Rhizopus microsporus strain into a substrate to form a fermented food product, the substrate comprising a legume, a legume processing residue, or a combination thereof.

[0007] According to an embodiment of the present disclosure, a fermented food product is provided. The fermented food product is formed by fermenting a substrate with an isolated and purified strain of Rhizopus microsporus. The accession number of the isolated and purified strain of Rhizopus microsporus is DSM 34400. The substrate includes a legume, a processing residue of a legume, or a combination thereof. Furthermore, the fermented food product has a hardness of 6.48N to 27.54N, a springiness of 0.24Nm to 0.70Nm, a chewiness of 0.70N to 5.00N, a gumminess of 2.7N to 10.05N, a cohesiveness of 0.32 to 0.80, and a resilience of 0.14 to 0.50.

[0008] The following embodiments will be described in detail with reference to the accompanying drawings. [Brief description of the drawings]

[0009] [Figure 1] 1A to 1C show a process of screening filamentous single strain Tp-1 from colonies in culture according to an embodiment of the present disclosure. [Diagram 2]1 shows an alignment of the sequence of strain Tp-1 (SEQ ID NO:1) with the sequence of Rhizopus microspores isolate TB55 using BLAST, according to an embodiment of the present disclosure. [Diagram 3] FIG. 1 shows a growth curve diagram (relationship between incubation time and area percentage) of strain Tp-1 during growth in PDA medium according to an embodiment of the present disclosure. [Figure 4] FIG. 1 shows the growth curves (relationship between incubation time and area percentage) of strain Tp-1 when grown on PDA medium with different proportions of bean dregs, according to an embodiment of the present disclosure. [Diagram 5] 1 shows a comparison of photographs taken before and after culturing strain Tp-1 in PDB medium with different proportions of bean dregs, according to embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The novel isolated and purified Rhizopus microsporus strain according to the present disclosure, the method for preparing fermented food using the Rhizopus microsporus strain, and the fermented food are described in detail in the following description. In order to fully understand the present disclosure, it should be understood that the following detailed description shows many characteristic details and embodiments for the purpose of explanation. The specific elements and configurations described in the following detailed description are shown to clearly explain the present disclosure. It should be clear that the exemplary embodiments shown herein are used for the purpose of explanation only, and do not limit the present disclosure.

[0011] In the following description, the terms "about" and "substantially" mean typically + / - 10% of the given value, or typically + / - 5% of the given value, or typically + / - 3% of the given value, or typically + / - 2% of the given value, or typically + / - 1% of the given value, or typically + / - 0.5% of the given value. The given values ​​in this disclosure are approximate values. Unless otherwise specified, the given values ​​are intended to include the meanings of "about" and "substantially". In addition, the terms "in the range from a first value to a second value" or "in the range from a first value to a second value" mean that the range includes the first value, the second value, and any other values ​​therebetween.

[0012] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In each case, it should be understood that terms defined in commonly used dictionaries should be interpreted to have a meaning consistent with the relevant art of this disclosure and the background or context of this disclosure, and should not be interpreted in an idealized or overly formal manner unless defined.

[0013] According to an embodiment of the present disclosure, a novel Rhizopus microsporus strain is isolated and purified from banana leaves of bananas planted in Qishan, Kaohsiung, Taiwan. The Rhizopus microsporus strain has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the accession number DSM 34400. The Rhizopus microsporus strain has also been deposited at the Food Industry Research and Development Institute under the accession number BCRC 930226. Furthermore, according to an embodiment of the present disclosure, by using legumes or their processing residues as a culture substrate and carrying out a fermentation process with the above-mentioned Rhizopus microsporus strain, the fermented food produced has a high protein content and is easily digestible in the human intestinal tract. The fermented food produced is also easily processed into a vegetable protein (TVP) additive. Moreover, the fermented foods can replace livestock, poultry and seafood as plant-based proteins.The fermented foods have the characteristics of low calories, low cholesterol and high quality protein, providing a diverse source of healthy foods and also contributing to a significant extent to net zero carbon dioxide emissions in the environment.

[0014] As mentioned above, a novel isolated and purified Rhizopus microsporus strain is provided in the present disclosure, and its accession numbers are DSM 34400 (deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on September 29, 2022) and BCRC 930226 (deposited at the Bioresource Collection and Research Center of the Food Industry Research and Development Institute on September 23, 2021). In detail, the strain was obtained from banana leaves in the banana-producing area of ​​Qishan, Kaohsiung, Taiwan through the steps of microbial screening, culture, and isolation and purification. The strain has been identified as Rhizopus microsporus TB55 strain. According to some embodiments, the nucleotide sequence of the isolated and purified novel Rhizopus microsporus strain has at least about 95% similarity to the sequence shown in SEQ ID NO:1, and may have, for example, 96%, 97%, 98% or 99% similarity. According to some embodiments, the nucleotide sequence of the isolated and purified novel Rhizopus microsporus strain is shown in SEQ ID NO:1.

[0015] In the present disclosure, a method for producing a fermented food product using a Rhizopus microsporus strain is provided, which includes the steps of: (a) preparing the above-mentioned isolated and purified Rhizopus microsporus strain having the deposit number DSM 34400 (BCRC 930226); and (b) inoculating a substrate with the isolated and purified Rhizopus microsporus strain to ferment and form a fermented food product. Furthermore, the substrate may include, but is not limited to, a legume, a legume processing residue, another suitable substrate, or a combination thereof.

[0016] According to some embodiments, the isolated and purified Rhizopus microsporus strain is provided in the form of a freeze-dried powder or bacterial broth.

[0017] According to some embodiments, legumes may include, but are not limited to, soybeans, black beans, adzuki beans, mung beans, edamame, kidney beans, red kidney beans, pinto beans, peas, pigeon peas, cowpeas, fava beans, chickpeas, lentils, hyacinth beans, peanuts, lupins, grass peas, carob, or combinations thereof.

[0018] According to some embodiments, legume processing residues may include, but are not limited to, legume dregs. According to some embodiments, legumes may be subjected to processing to obtain the above-mentioned processing residues, which may include, but are not limited to, a cooking step, a pressing step, a drying step, a grinding step, or a combination thereof. According to some embodiments, the moisture content of the processing residues may be less than about 15%, such as less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5%.

[0019] According to some embodiments, the substrate used for fermentation can be legume residue, i.e. soybean lees (okara). Soybean lees is a by-product of bean processing, which usually produces a large amount of soybean lees. Soybean lees contain many functional ingredients, such as isoflavones, phenolic acids, and phytosterols. However, soybean lees are usually only discarded or used as feed or fertilizer due to their high water content during production, which may make them prone to spoilage and odor. In particular, according to embodiments of the present disclosure, soybean lees can be reused in a circular economy manner, and the protein content and nutritional value of soybean lees can be increased by the fermentation process, and the amount of soybean lees that is discarded can be reduced, reducing the burden on the environment.

[0020] According to some embodiments, the fermentation of the Rhizopus microsporus strain in the substrate may include a solid fermentation process, a liquid fermentation process, or a combination thereof. According to some embodiments, the temperature of the fermentation may be about 20° C. to about 40° C., such as 25° C., 30° C., or 35° C. According to some embodiments, the fermentation may be performed for about 20 hours to about 50 hours, or about 24 hours to about 48 hours, such as 28 hours, 32 hours, 36 hours, 40 hours, or 44 hours.

[0021] According to some embodiments, the isolated and purified Rhizopus microsporus strain can be inoculated into a substrate for fermentation, and the substrate can be further subjected to a uniaxial wet extrusion process or a biaxial wet extrusion process to form a fermented food product. According to some embodiments, the fermented food product produced by the above method can include, but is not limited to, tempeh, vegetable protein (TVP) additives, functional beverages, or combinations thereof. According to some embodiments, the fermentation broth produced by the fermentation can be directly used as a functional beverage.

[0022] Additionally, embodiments of the present disclosure also provide a fermented food product. The fermented food product is formed by fermenting a substrate with the isolated and purified Rhizopus microsporus strain described above. The hardness of the fermented food product may be about 6.48N to about 27.54N. The springiness of the fermented food product may be about 0.24Nm to about 0.70Nm. The chewiness of the fermented food product may be about 0.7N to about 5.00N. The gumminess of the fermented food product may be about 2.7N to about 10.05N. The cohesiveness of the fermented food product may be about 0.32 to about 0.80. The resilience of the fermented food product may be about 0.14 to about 0.50. According to some embodiments, the above parameters such as hardness, springiness, chewiness, gumminess, cohesiveness and resilience are measurements obtained by a Texture Analyzer (Horn Instruments co., ltd., Model: UniversalTA).

[0023] As discussed above, the substrate may include, but is not limited to, a legume, legume processing residues, another suitable substrate, or a combination thereof.

[0024] According to some embodiments, legumes may include, but are not limited to, soybeans, black beans, adzuki beans, mung beans, edamame, kidney beans, red kidney beans, pinto beans, peas, pigeon peas, cowpeas, fava beans, chickpeas, lentils, hyacinth beans, peanuts, lupins, grass peas, carob, or combinations thereof.

[0025] According to some embodiments, legume processing residues may include, but are not limited to, legume dregs.Further, according to some embodiments, fermented foods may include, but are not limited to, tempeh, vegetable protein (TVP) additives, functional beverages, or combinations thereof.

[0026] It should be noted that the fermented food produced by the method of producing fermented food using Rhizopus microsporus strains provided by the embodiments of the present disclosure has an increased content of protein and water-soluble dietary fiber, and can regulate blood sugar, lower cholesterol, delay sugar absorption in the small intestine, and strengthen intestinal peristalsis. In addition, the resulting fermented food (such as tempeh) can improve the digestion and absorption rate of protein, is easy to digest, and is less likely to cause flatulence, so as to meet the protein requirements of the elderly, the infirm, and those with weak digestive systems.

[0027] Specifically, in some embodiments, the resulting fermented food may have a protein content that is increased by about 2% to about 10% compared to the unfermented substrate. Specifically, in some embodiments, the resulting fermented food may have a protein content that is increased by about 22% to about 28% by total weight of the fermented food. In some embodiments, the resulting fermented food may have a soluble dietary fiber content that is increased by about 2.70% to about 5.67% by total weight of the fermented food.

[0028] In order to make the above and other objects, features and advantages of the present disclosure more fully and easily understood, some examples and comparative examples are presented and described in detail below, but they are not intended to limit the scope of the present disclosure.

[0029] Example 1: Isolation, purification and identification of a novel Rhizopus microsporus strain

[0030] Banana leaves planted in Qishan area, Kaohsiung, Taiwan were collected. After washing and drying, the banana leaves were used to cover the dehulled soybeans for fermentation and growth. The filamentous fungi attached to the banana leaves were then cultured, screened, isolated, purified and identified.

[0031] First, 100 μl of broth (potato dextrose broth, STBIO MEDIA, INC.) used for culturing filamentous fungi was taken, 900 μl of secondary water was added, and the mixture was spread on a PDA medium plate (potato dextrose agar broth; Becton, Dickinson and Company, USA) and cultured at 30°C for 24 hours. Next, a colony on the medium was picked and streaked again on the same PDA medium plate to purify the strain. After culturing at 30°C for 24 hours, the mycelium was picked, stained using the Gram staining method, and observed under an electron microscope. Screening was repeated several rounds until a single-phase strain was observed under the electron microscope. See Figures 1A to 1C. These show the process of screening a single filamentous strain (strain number Tp-1) from the colonies on the medium. Then, 20% glycerol was added to the isolated and purified strain, which was then frozen and stored at -80°C, and a portion of the strain was taken for subsequent nucleic acid identification.

[0032] The DNA of the above-mentioned strains was extracted according to the instructions of the QIAGEN DNeasy Plant Kit. The fungal universal primer pair ITS4 (shown in SEQ ID NO:2) and ITS5 (shown in SEQ ID NO:3) (MISSION BIOTECH CO., LTD.) was used to sequence the internal transcribed spacer (ITS) by polymerase chain reaction. The DNA extracted sample of strain Tp-1 was sequenced using the Applied Biosystem® (ABI) BigDye Terminator v3.1 Cycle Sequencing Kit and a sequencing machine ABI 3730XL DNA Analyzer, and the obtained nucleotide sequence is shown in SEQ ID NO:1.

[0033] The nucleotide sequence of SEQ ID NO:1 was then aligned with known biological sequence databases (National Center for Biotechnology Information, NCBI) using BLAST (Basic Local Alignment Search Tool). This program aligns nucleotide or protein sequences with sequence databases, calculates statistical significance, and combines validation of physiological and biochemical characteristics. It was confirmed that the above-mentioned isolated and purified strain Tp-1 was more than 99% similar to the Rhizopus microspores isolate TB55 in the database.

[0034] The sequence alignment result between SEQ ID NO:1 and Rhizopus microspores isolate TB55 (gene bank: MF445290.1) is shown in Figure 2. The sequence read length of TB55 is 695 bp, of which 660 bp in the sequence is the same as SEQ ID NO:1, and 35 bp in the sequence is different from SEQ ID NO:1. Therefore, it can be determined that the isolated and purified strain Tp-1 belongs to Rhizopus microsporus strain TB55, but Tp-1 and TB55 are different strains.

[0035] After identification was completed, the isolated and purified novel Rhizopus microsporus strain Tp-1 was deposited and preserved at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 344006. The isolated and purified novel Rhizopus microsporus strain Tp-1 was also deposited and preserved at the Bioresource Conservation and Research Center of the Institute for Food Industry Development under the accession number BCRC 930226.

[0036] Example 2: Growth curve plot and analysis of novel Rhizopus microsporus strains

[0037] Using an inoculation loop, a suitable amount of mold spores was collected from the Tp-1 strain sample onto a PDA medium plate (Difco® Potato Dextrose Agar), incubated at 30°C, and photographed at regular intervals (8 to 12 hours). Rhizopus has a filamentous morphology, so it grows in a staggered and irregular shape on the solid PDA medium plate. Therefore, an area percentage calculation was used for quantitative analysis, and a growth curve was plotted using the relationship between the area percentage of the growing strain and time. The life cycle of the Tp-1 strain in the fermentation process can be known, allowing the subsequent fermentation conditions (e.g., fermentation time) to be evaluated.

[0038] Please refer to Figure 3, which shows the growth curve diagram (relationship between incubation time and area percentage) of strain Tp-1 during growth on PDA medium plate. As shown in Figure 3, the growth rate of strain Tp-1 began to increase about 30 hours after incubation, and the period from about 34 to 49 hours of incubation was the rapid growth period of strain Tp-1. About 50 to 57 hours after incubation, the growth rate gradually slowed down, the area percentage of Tp-1 growing on the medium plate exceeded 90%, and the distribution of white mycelium in the medium approached saturation. The growth analysis of strain Tp-1 shows that strain Tp-1 is suitable for the production of solid fermentation process.

[0039] Example 3: Growth and functionality test analysis of novel Rhizopus microsporus strains with the addition of bean processing residues

[0040] Using an inoculation loop, appropriate amounts of mold spores were collected from the Tp-1 strain samples onto PDA medium plates (Difco® Potato Dextrose Agar) containing 0%, 5%, 6% and 7% (w / v%) soybean meal, respectively, and incubated at 30°C and photographed at regular intervals (8 to 12 hours). Area percentage calculation method was used for quantitative analysis, and growth curves were plotted using the relationship between the area percentage of the growing strain and time.

[0041] Please refer to Figure 4, which shows the growth curves (relationship between incubation time and area percentage) of strain Tp-1 when growing on PDA medium plates containing different proportions of soybean residue. As shown in Figure 4, the overall growth trend of strain Tp-1 in the medium containing soybean residue was better than that in the medium without soybean residue, and the growth rate of strain Tp-1 in the medium containing soybean residue was faster. In addition, the growth rate of strain Tp-1 in the medium containing 6% soybean residue was the fastest, while the growth rates of strain Tp-1 in the medium containing 5% soybean residue and 7% soybean residue were similar at all stages. From the above results, it can be seen that strain Tp-1 prefers a medium with a higher protein content.

[0042] Example 4: Analysis of components of fermentation products of a novel Rhizopus microsporus strain

[0043] Using a platinum loop, appropriate amounts of mold spores were collected from Tp-1 strain samples into PDB broth (Potato Dextrose Broth, STBIOMEDIA) containing 0%, 5%, 6% and 7% (w / v%) soybean meal, respectively. Fermentation culture was carried out at 30°C and 120 rpm for 4 days. After the culture was completed, the medium was centrifuged at 20°C and 9900 rpm for 40 minutes, and the supernatant and precipitate were stored separately. The precipitate was then freeze-dried for 2 days and ground into powder, and the fermentation products were analyzed for the contents of crude protein, crude fat, carbohydrate, soluble dietary fiber and insoluble dietary fiber. The measurement methods were as follows. The experimental results are shown in Table 1.

[0044] 1. Analysis of Crude Protein Content

[0045] The total nitrogen content was determined using the Kjeldahl nitrogen method. 0.5 g of the sample was taken and mixed with 5 g of catalyst (K 2 SO 4 :CuSO 4 H 2The digestion tube was then placed in a protein digestion furnace and heated to 200°C for 1 hour. The sample was carbonized to a black liquid, then heated to 400°C for 2 hours. After digestion to a clear light blue state, it was taken out and cooled, and then 70 mL of distilled water and 50 mL of 40% NaOH were added. Nitrogen was distilled using a nitrogen distillation apparatus, and the distilled nitrogen was received with 4% boric acid water containing methyl blue and methyl red indicators, turning the liquid red. Finally, the liquid was titrated with 0.1N HCl until the liquid turned slightly reddish (i.e., the titration was complete), and the amount of the titrated liquid was recorded. Crude protein content (%) = [HCl titration amount (ml) x HCl concentration (N) x 1.4007 x 1] / sample weight (g) x nitrogen content factor (nitrogen conversion factor) 6.25.

[0046] 2. Analysis of crude fat content

[0047] 3 g of sample was taken on filter paper, wrapped and then placed in a filter thimble. Then, it was placed in a Soxhlet extractor connected to a fat collection bottle, and 170 mL of n-hexane was added from the top of the extractor. The extractor was connected to a condenser tube and refluxed at 50-60°C in a constant temperature water bath for 16 hours. After extraction, the fat collection bottle was removed and the n-hexane in the Soxhlet extractor was collected. The fat collection bottle was placed in a 105°C oven to dry, removed and placed in a drying dish, and cooled until the fat collection bottle was at constant weight. Crude fat (%) = [(weight of fat collection bottle after fat extraction - weight of fat collection bottle)] / weight of sample x 100%.

[0048] 3. Analysis of total carbohydrate content

[0049] Total carbohydrate content is obtained by subtracting the percentages of moisture, ash, crude protein and crude fat from 100%.

[0050] 4. Analysis of soluble dietary fiber content

[0051] The insoluble dietary fiber was separated from the filtrate by the above-mentioned extraction process, and the soluble dietary fiber was precipitated by pouring the filtrate into a beaker containing 300 mL of 95% alcohol and leaving it to settle for one day to allow complete precipitation. The weight of the filter paper was weighed and recorded to four decimal places. The liquid was poured into a magnetic funnel and filtered by suction, rinsed three times with 20 mL of 78% alcohol, then rinsed twice with 10 mL of 95% alcohol, and finally rinsed twice with 10 mL of acetone. The filter paper and residue were placed together in a weighing bottle and dried in an oven at 105 °C until constant weight was reached, and the weight of the weighing bottle and filter paper were subtracted from the constant weight to obtain the total residue weight. Water-soluble dietary fiber content (%) = [total residue net weight - weight of protein in sample (g) - weight of ash in sample (g)] / weight of sample (g) x 100%.

[0052] 5. Analysis of insoluble dietary fiber content

[0053] A thermostatic water bath was preheated to 95°C, and approximately 1 g of powder sample was weighed out and recorded to 4 decimal places. The sample was placed in a beaker, 50 mL of phosphate buffer was added, and mixed uniformly. 100 μl of α-amylase was added and gently shaken. The beaker was covered with aluminum foil and placed in a thermostatic water bath at 95°C for approximately 15 minutes, gently shaken every 5 minutes, cooled to room temperature, and then 10 mL of 0.275N NaOH was added and gently shaken. A thermostatic water bath was preheated to 60°C, and 0.05 g of protease was added to 1 mL of phosphate buffer, mixed uniformly, and 100 μl of protease phosphate buffer solution was added to the beaker. The beaker was covered with aluminum foil and placed in a thermostatic water bath at 60°C, gently shaken every 5 minutes for approximately 30 minutes, cooled to room temperature, and then 10 mL of 0.325N HCl was added and gently shaken. A thermostatic water bath was preheated to 60°C, and 100 μl of amyloglucosidase was added to the beaker. The beaker was covered with aluminum foil and placed in a thermostatic water bath at 60°C, gently shaking every 5 minutes for approximately 30 minutes, and then cooled to room temperature. The filter paper was weighed and recorded to four decimal places, and the liquid was poured into a magnetic funnel for suction filtration. The filter paper and residue were placed together in a weighing bottle and placed in a 105°C oven to dry to a constant weight, and the weight of the weighing bottle and filter paper were subtracted from the constant weight to obtain the total residue weight. Insoluble dietary fiber content (%) = [total residue net weight - weight of protein in sample (g) - weight of ash in sample (g)] / weight of sample (g) x 100%.

[0054] [Table 1]

[0055] According to the results in Table 1, after Tp-1 was inoculated into the medium containing different ratios of soybean residue (0%, 5%, 6%, 7% (w / v%)) and fermented for 96 hours, the crude protein content of the medium containing soybean residue increased. Compared with the group without soybean residue, the group containing 7% soybean residue had an increase of about 4% in protein content, about 6% in glycolysis conversion rate, and about 2.1% in soluble dietary fiber.

[0056] Comparative Example 1: Component analysis of fermentation products of BCRC 31750 tempeh fungus

[0057] A comparative experiment was carried out using the tempeh fungus with the accession number BCRC 31750. Using an inoculation loop, the tempeh fungus BCRC 31750 was inoculated into PDB broth (Potato Dextrose Broth, STBIO MEDIA) containing 0%, 5%, 6% and 7% (w / v%) soybean meal, respectively. Fermentation was carried out at 30°C and 120 rpm for 4 days. Figure 5 shows a comparison of photographs taken before and after the cultivation of strain Tp-1 in PDB medium containing different proportions of soybean meal.

[0058] In addition, after the cultivation was completed, the medium was centrifuged at 20°C and 9900 rpm for 40 minutes, and the supernatant and precipitate were stored separately. The precipitate was freeze-dried for 2 days and ground into powder, and the contents of crude protein, crude fat, carbohydrate, soluble dietary fiber and insoluble dietary fiber of the fermentation product were analyzed. The measurement method was as described in Example 4. The experimental results are shown in Table 2.

[0059] [Table 2]

[0060] According to the results in Table 2, after BCRC 31750 tempeh bacteria was inoculated into the medium containing different ratios of soybean residue (0%, 5%, 6%, 7% (w / v%)) and fermented for 96 hours, the crude protein content of the medium containing soybean residue did not increase significantly. Compared with the group without soybean residue, the glycolysis in the group containing soybean residue was not significant, and the soluble dietary fiber increased slightly by about 1.6%. Comparing Table 1 and Table 2, it can be seen that compared with BCRC 31750 tempeh bacteria, the novel Rhizopus microsporus strain Tp-1 provided by the present disclosure has the effect of increasing the protein and soluble dietary fiber content of the fermentation product, and the effect of increasing the glycolysis conversion rate.

[0061] Example 5: Physical properties of fermentation products of novel Rhizopus microsporus strains

[0062] Strain Tp-1 was inoculated into different soybean dregs and solid-state fermentation was carried out to produce soybean dreg tempeh, whose physical properties were measured by a texture analyzer (Horn Instruments co., ltd., model: UniversalTA). The soybeans used as the source for soybean dreg tempeh A, B and E were provided by Taiwan Taoyuan Tofu Association (imported soybeans), and the soybean used as the source for soybean dreg tempeh X was provided by PEACE VEGAN CO., LTD (Taiwanese organic soybeans).

[0063] The soybean residue from different sources mentioned above was subjected to a solid-state fermentation process. First, 1 kg of soybean residue was taken and dried in an oven at 60°C to 65°C for 24 hours and sterilized with steam. After cooling to room temperature, about 10 to 20 grams of Tp-1 strain powder or liquid was sprinkled on the soybean residue and stirred evenly before being placed in a fermentation chamber at 30°C, and the fermented product was obtained after 24 to 48 hours. The fermented product was extracted from 2 cm 3 A square of each was taken as a sample and subjected to texture analyzer measurements to analyze its physical properties such as hardness, springiness, chewiness, gumminess, cohesiveness and resilience.

[0064] The texture analyzer data of soybean residue tempeh A (sample 1), soybean residue tempeh B (sample 2), soybean residue tempeh E (sample 3) and soybean residue tempeh X (sample 4) from different sources prepared as above are shown in Table 3. Furthermore, the data were compared with those of commercial soybean tempeh (purchased from XiangHehJia-Biotech CO., LTD.) (sample 5), commercial wheat TVP (provided by GOLDENCROPS CORPORATION) (sample 6) and commercial pea TVP (provided by GOLDENCROPS CORPORATION) (sample 7).

[0065] [Table 3]

[0066] As shown in the results in Table 3, the fermentation product (soybean meal tempeh) produced by fermentation of strain Tp-1 had a hardness comparable to that of pea TVP, and other parameter values ​​were lower. It can be seen that the texture of soybean meal tempeh is relatively soft and easy to chew. With suitable seasoning and simple hygienic processing, it can be used as a ready-to-eat conditioning package, which is very suitable for providing high-protein nutrition to patients or elderly people who need medical care. In vegetable meat products and vegetarian food semi-finished products, soybean meal tempeh can also be used as a natural protein source or ingredient additive to enhance the variety or flavor of food ingredients.

[0067] Although some embodiments of the present disclosure and their advantages have been described above, it should be understood that various changes, substitutions and alterations can be made thereto without departing from the spirit and scope of the present disclosure as defined by the appended claims. In addition, each claim constitutes an independent embodiment, and the claims of the present disclosure may also include combinations of claims and embodiments. The scope of protection of the present disclosure is subject to the definition of the appended claims.

Claims

1. A novel isolated and purified strain of Rhizopus microsporus having the deposit number DSM 34400.

2. A method for producing a fermented food using a Rhizopus microsporus strain, comprising: Providing an isolated and purified strain of Rhizopus microsporus having the deposit number DSM 34400; inoculating the isolated and purified Rhizopus microsporus strain into a substrate to ferment and form a fermented food; Including, The method, wherein the substrate comprises a legume, a legume processing residue, or a combination thereof.

3. The method of claim 2 , wherein the substrate is the processing residue of the legume.

4. 4. The method of claim 3, wherein the legume is subjected to processing to obtain the processing residue of the legume, the processing comprising a cooking step, a pressing step, a drying step, a grinding step, or a combination thereof.

5. 4. The method of claim 3, wherein the legume processing residues include dregs of the legume.

6. 3. The method of claim 2, wherein the legumes include soybeans, black beans, adzuki beans, mung beans, edamame, kidney beans, red kidney beans, pinto beans, peas, pigeon peas, cowpeas, fava beans, chickpeas, lentils, hyacinth beans, peanuts, lupins, grass peas, carob, or combinations thereof.

7. 3. The method of claim 2, wherein the fermentation comprises a solid fermentation process, a liquid fermentation process or a combination thereof, the temperature of the fermentation is between 20° C. and 40° C., and the fermentation is carried out for between 20 hours and 50 hours.

8. 3. The method of claim 2, wherein the isolated and purified Rhizopus microsporus strain is provided in the form of a freeze-dried powder or bacterial broth.

9. The method of claim 2, wherein the fermented food product comprises tempeh, a vegetable-based protein (TVP) additive, or a functional beverage.

10. After inoculating the substrate with the isolated and purified Rhizopus microsporus strain and carrying out fermentation, the method further comprises subjecting the substrate to a uniaxial wet extrusion process or 3. The method of claim 2, further comprising performing a biaxial wet extrusion process to form the fermented food product.

11. A fermented food product formed by fermenting a substrate using an isolated and purified strain of Rhizopus microsporus, the isolated and purified strain of Rhizopus microsporus having a deposit number of DSM 34400; the substrate comprises a legume, a legume processing residue, or a combination thereof; the fermented food product has a hardness of 6.48N to 27.54N, or a springiness of 0.24Nm to 0.70Nm, or a chewiness of 0.70N to 5.00N, or a gumminess of 2.7N to 10.05N, or a cohesiveness of 0.32 to 0.80, or a resilience of 0.14 to 0.50, The protein content of the fermented food is increased by 2% to 10% compared to the protein content of the unfermented substrate, the protein content of the fermented food is 22% to 28%, and the soluble dietary fiber content of the fermented food is 2.70% to 5.67%. Fermented foods.

12. 12. The fermented food product of claim 11, wherein the residues of processing of the legumes include dregs of the legumes.

13. 12. The fermented food product of claim 11, wherein the legumes include soybeans, black beans, adzuki beans, mung beans, edamame, kidney beans, red kidney beans, pinto beans, peas, pigeon peas, cowpeas, fava beans, chickpeas, lentils, hyacinth beans, peanuts, lupins, grass peas, carob, or combinations thereof.

14. The fermented food product of claim 11 , wherein the fermented food product comprises tempeh, a vegetable protein (TVP) additive, or a functional beverage.

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