A strain of Bacillus coagulans and its application

Abstract

The present invention discloses a strain of Bacillus coagulans, which is classified and named as Bacillus coagulans ( Bacillus coagulans ) PR06, which has been deposited in the China Center for Type Culture Collection on December 10, 2021, with the deposit number: CCTCC NO: M 20211589. The Bacillus coagulans PR06 provided by the present invention has strong acid and bile resistance, and has no resistance to most antibiotics. The present invention also discloses the use of the Bacillus coagulans in the preparation of fermented rice vermicelli or fermented rice vermicelli products. The fermented tea rice vermicelli prepared by using the Bacillus coagulans PR06 provided by the present invention has no bitter taste, excellent cooking and sensory quality, and pleasant flavor. Therefore, the Bacillus coagulans PR06 provided by the present invention has a high social application value.

Description

Technical Field

[0001] The invention relates to the technical field of microbial strains and their applications, and in particular to a Bacillus coagulans strain and its application. Background Art

[0002] Bacillus coagulans belongs to the genus Bacillus of the Firmicutes phylum. Its cell body is rod-shaped, its spores are terminal and it has no flagella. It is a facultative anaerobic Gram-positive bacterium. Bacillus coagulans can grow in both aerobic and anaerobic environments, can adapt to the hypoxic intestinal environment, has a high tolerance to acid and bile, can carry out lactic acid fermentation, and the L-lactic acid produced can reduce the pH value of the intestine, inhibit harmful bacteria, and promote the growth and reproduction of beneficial bacteria such as bifidobacteria. Bacillus coagulans can form spores, which is beneficial to restore the microecological balance of the gastrointestinal tract compared with other Bacillus that do not produce lactic acid. Bacillus coagulans spores can germinate in the human body in about 4-6 hours, and 85% of the bacteria can pass through the digestive system smoothly, and finally germinate and reproduce in the intestine. Compared with the important lactobacillus and bifidobacteria inherent in the gastrointestinal tract, Bacillus coagulans not only has the dual effects of the two, but also has the unique advantages of Bacillus such as high temperature resistance, easy cultivation and storage stability. Studies have shown that Bacillus coagulans can improve the body's immunity, enhance disease resistance, and promote the body's digestion and metabolism and the absorption of nutrients. At present, Bacillus coagulans has been listed by the Ministry of Agriculture of China as a new feed additive that can be directly used in livestock and poultry production. Bacillus coagulans is one of the strains approved by my country for use in ordinary food, as stated in Announcement No. 6 of 2014 by the National Health and Family Planning Commission of the People's Republic of China. In the food field, Bacillus coagulans are mainly used in probiotic soft candies and room temperature acidic drinks. At present, there are no reports in the industry on the application of Bacillus coagulans in fermented tea rice vermicelli.

[0003] Rice noodles are a common staple food in southern my country, with hundreds of millions of people eating them. It is the rice product with the largest market size. In 2019, the annual output value of Changde rice noodles and Guilin rice noodles in my country exceeded 2 billion and 2.5 billion yuan respectively. Rice noodles can be divided into fermented rice noodles and non-fermented rice noodles. Studies have shown that fermented rice noodles have greatly improved taste and flavor, and have better cooking characteristics and rehydration properties. Since the nutritional components of ordinary rice noodles are relatively simple (about 75% starch and about 7% protein), they lack health functional ingredients. In recent years, with the enhancement of people's health awareness, ordinary rice noodles can no longer meet people's requirements for nutrition and health. Tea is known as the "national drink" in my country. The health benefits of tea have long been accepted by the public. The health ingredients mainly include tea polyphenols and theanine. Tea food meets consumers' expectations and requirements for health care. The application of tea to various foods to process them into foods with tea characteristics and health benefits is increasingly favored by consumers. Since the starch in rice noodles is relatively easy to be digested and absorbed by the human body, it is easy to cause an increase in blood sugar levels in the human body after consumption, which is not suitable for diabetic patients. Studies have shown that polyphenols can complex with starch macromolecules, change the crystal form of starch molecules, and then affect the aging behavior and digestion properties of starch gel, playing an anti-digestive role. Therefore, adding tea powder containing polyphenols during the rice flour processing can delay the digestion of rice flour and is suitable for patients with high blood sugar. However, tea polyphenols and caffeine in tea can cause rice flour to have a certain bitter taste.

[0004] To solve the above problems, the applicant is committed to screening out strains for preparing fermented tea rice vermicelli or rice noodle strips without bitterness, providing a new source of bacteria for the development and production of fermented tea rice vermicelli or rice noodle products. Summary of the invention

[0005] The purpose of the present invention is to provide a Bacillus coagulans strain and application thereof, and to develop a strain that can be used for preparing fermented tea rice vermicelli (noodles) without bitterness.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A strain of Bacillus coagulans, the strain is classified and named Bacillus coagulans PR06, which was deposited in the China Center for Type Culture Collection on December 10, 2021, with the deposit number: CCTCC NO: M20211589, the address of the deposit unit: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Postal Code: 430072.

[0008] The Bacillus coagulans PR06 provided by the present invention is separated from human feces, and the colony characteristics are as follows: after being cultured on an MRS plate culture medium for 24 to 48 hours, the colony morphology is milky white, round, with a smooth surface, moist and shiny; scanning electron microscope observation shows that the bacteria are rod-shaped, with a size of 0.2 to 0.4×2.0 to 5.0 μm; and the optimal growth temperature is 50°C.

[0009] The present invention also provides the use of the Bacillus coagulans PR06 in the processing and production of common fermented rice vermicelli or common fermented rice noodle strips.

[0010] The present invention also provides the use of the Bacillus coagulans PR06 in the processing and production of fermented tea rice vermicelli or fermented tea rice vermicelli strips.

[0011] The significant advantages of the present invention are:

[0012] The Bacillus coagulans PR06 provided by the present invention has strong acid and bile salt resistance and is not resistant to most antibiotics. The common fermented rice vermicelli prepared by using the Bacillus coagulans PR06 provided by the present invention has excellent sensory quality, unique and pleasant flavor. The fermented tea rice vermicelli prepared by using the Bacillus coagulans PR06 provided by the present invention has no bitter taste, excellent cooking and sensory quality, and pleasant flavor. Therefore, the Bacillus coagulans PR06 provided by the present invention has a high social application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the colony morphology of Bacillus coagulans PR06.

[0014] Figure 2 This is a scanning electron micrograph of Bacillus coagulans PR06.

[0015] Figure 3 This is the growth curve of Bacillus coagulans PR06 at different temperatures.

[0016] Figure 4 This is the finished product of tea rice vermicelli fermented by Bacillus coagulans PR06 (before steaming).

[0017] Figure 5 This is the finished product of tea rice vermicelli fermented with Bacillus coagulans PR06 (after steaming). DETAILED DESCRIPTION

[0018] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, the culture medium and experimental conditions in the examples are conventional culture medium and experimental conditions. The experimental materials, reagents, etc. used in the examples, unless otherwise specified, can all be obtained from commercial sources.

[0019] Example 1 Isolation and Identification of Bacillus coagulans PR06

[0020] 1. Strain Isolation

[0021] 1.0 g of fresh human feces sample was taken and placed in a sterile centrifuge tube containing 9.0 mL of sterile water. The mixture was shaken and mixed. The obtained bacterial suspension was placed in a water bath at 80 °C boiling water for 30 min and then taken out. Then, the suspension was diluted in sequence using the 10-fold gradient dilution method. 100 μL of dilution solution with an appropriate concentration gradient was selected and added to MRS agar (tryptone 10.0 g / L, beef powder 5.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, K2HPO4·3H2O 1.5 g / L, anhydrous sodium acetate 3.0 g / L, ammonium citrate 2.0 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·H2O 0.04g / L, agar powder 20.0g / L, distilled water 1L, pH value 6.2-6.4) plate, spread evenly with a sterile coating rod, use sterile water as blank control, and place inverted culture in a 45℃ constant temperature incubator for 48-72h after spreading evenly. Pick white or off-white, shiny, round, with neat edges, and colony strains, and repeatedly streak and purify them on MRS agar plate culture medium until a single colony is obtained, which is named PR06.

[0022] 2. Strain identification

[0023] (1) Observation of colony and bacterial morphology: Strain PR06 was streaked on MRS agar plate and cultured at 37°C for 24-48 h. Figure 1 ) is milky white, round, smooth, moist and shiny. Under a scanning electron microscope, the bacteria are rod-shaped, with a size of 0.2-0.4 μm × 2.0-5.0 μm. Figure 2 .

[0024] (2) Observation of the growth characteristics of strain PR06: The bacterial suspension of strain PR06 was inoculated into MRS liquid culture medium at a volume ratio of 3.0% and cultured at different temperatures for 24 h. It was found that the strain grew well in an environment of 30°C to 60°C, and the optimal growth temperature was 50°C (see Figure 3 ).

[0025] (3) Molecular biological identification of strain PR06: The whole genome of strain PR06 was extracted using a bacterial whole genome rapid extraction kit, and PCR was performed by selecting bacterial 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'), followed by sequencing analysis. The sequencing results were compared by BLAST, and the strain was identified as Bacillus coagulans. The gene sequence is shown in SEQ ID NO.1, and it was named: Bacillus coagulans PR06, which was deposited in the China Center for Type Culture Collection on December 10, 2021, with the deposit number: CCTCC NO: M 20211589. The address of the deposit unit: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Postal Code: 430072.

[0026] Example 2 Utilization of carbohydrates by Bacillus coagulans PR06 The API50CHL kit (Biomerieux, France) was used to detect the utilization of carbohydrates by the strain PR06 isolated in Example 1. The results (Table 1) showed that the Bacillus coagulans PR06 of the present invention could utilize ribose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetylglucosamine, ferric citrate of esculin, D-maltose, D-lactose, D-melanobiose, D-sucrose, trehalose, D-raffinose and starch.

[0027] Table 1 Results of using API50CHL kit to detect the utilization of sugars by strain PR06

[0028] Substrate ingredients 48h reaction results Substrate ingredients 48h reaction results Comparison - Esculin Ferric Citrate + glycerin - Salicin (Salicin) - Erythritol - D-Cellobiose - D-arabinose - D-Maltose + L-arabinose - D-Lactose + Ribose + D-Mesbiose + D-Xylose - D-sucrose + L-Xylose - Trehalose + Adonit - Inulin (Inulin) - β-Methyl-D-xyloside - D-Melezitose - D-Galactose + D-Raffinose + D-Glucose + starch + D-Fructose + Glycogen - D-Mannose + Xylitol - L-sorbitol - D-gentiobiose - L-Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol - D-Tagatose - Mannitol + D-fucose (D-rock sugar) - Sorbitol + L-fucose - α-Methyl-D-pyranose - D-arabinol (D-arabitol) - α-Methyl-D-glucopyranoside - L-Arabitol - N-acetyl glucosamine + Potassium Gluconate (Salt) - Amygdalin - 2-Ketogluconate Potassium Salt - ARBULIN - 5-Ketogluconate Potassium Salt -

[0029] Note: + indicates a positive result, indicating that the strain can utilize the tested substrate; - indicates a negative result, indicating that the strain cannot utilize the tested substrate.

[0030] Example 3 Acid and bile resistance of Bacillus coagulans PR06

[0031] The activated strain PR06 was made into a suspension, and 1.0 mL of bacterial suspension (concentration of 2.0×10 7 cfu / mL) were inoculated into 9.0 mL of MRS liquid culture medium with a pH of 2.5, 3.0, 3.5 or a bile salt concentration of 1.0, 2.0, 3.0 and 4.0 g / L, respectively, and cultured at 45°C for 4 h. The number of viable bacteria was determined by plate count method at 0 h and 4 h after culture, and the survival rate (%) was calculated.

[0032] Survival rate (%) = (number of viable bacteria at 4 h / number of viable bacteria at 0 h) × 100.

[0033] Table 2 Acid and bile salt resistance of Bacillus coagulans PR06

[0034]

[0035] The results in Table 2 show that Bacillus coagulans PR06 has a high survival rate in an environment with a pH of 3.0, 3.5 or a bile salt concentration of 1.0, 2.0, 3.0 and 4.0 g / L, indicating that the strain has strong acid and bile salt resistance and can successfully pass through the gastrointestinal tract into the large intestine of the body to exert a probiotic effect.

[0036] Example 4 Antibiotic resistance of Bacillus coagulans PR06 PR06 bacterial solution was inoculated into MRS liquid culture medium, cultured in a 45°C biochemical incubator for 24 hours, 100 μL of bacterial solution was spread on an MRS agar plate, and tablets were placed thereon, the tablets containing erythromycin, penicillin, gentamicin, kanamycin, streptomycin, bacitracin, chloramphenicol, vancomycin, tetracycline, ampicillin, cefoxitin, rifampicin, neomycin, nalidixic acid, ciprofloxacin, polymyxin, and inverted cultured in a 45°C biochemical incubator for 24 hours, and the size of the inhibition zone was observed and recorded. The experimental results are shown in Table 3.

[0037] Table 3 Antibacterial effect of antibiotics on Bacillus coagulans PR06

[0038]

[0039]

[0040] The results in Table 3 show that Bacillus coagulans PR06 is resistant only to two antibiotics, bacitracin and polymyxin, and has no resistance to most other antibiotics, indicating that PR06 has good application safety.

[0041] Example 5 Preparation and sensory evaluation of rice vermicelli fermented with Bacillus coagulans PR06 Early indica rice with an amylose content of more than 20% and aged for 1 to 1.5 years was ground and passed through an 80-mesh sieve to obtain rice flour. 800 g of rice flour sample was added with 150 g of corn starch and 50 g of potato starch. After fully mixing, the rice flour was spread under an ultraviolet lamp for sterilization for 24 h. An appropriate amount of pure water was added to make the moisture content of the rice slurry 55% to 60%. The rice slurry was inoculated with activated Bacillus coagulans PR06 (concentration: 2.0×10 7cfu / mL, inoculation amount 2% v / v), after fermentation at 30-50°C for 8-18h, the fermented rice slurry is poured into a self-cooking extrusion rice noodle machine, the obtained vermicelli is steamed for 2-10min, cooled, aged at 4-8°C for 3-10h, and finally dried to a moisture content of less than 14% to obtain a finished product. The finished vermicelli was subjected to sensory evaluation, and the results showed that the sensory quality of the ordinary fermented rice vermicelli with Bacillus coagulans PR06 was excellent, with a pleasant sweet and sour taste when chewed.

[0042] Example 6 Preparation of tea rice vermicelli fermented with Bacillus coagulans PR06

[0043] Early indica rice with an amylose content of more than 20% and aged for 1 to 1.5 years was ground and passed through an 80-mesh sieve to obtain rice flour. 800 g of rice flour sample was added with 150 g of corn starch and 50 g of potato starch. After fully mixing, the mixture was spread out under ultraviolet light for sterilization for 24 h. An appropriate amount of tea was added (50 g of rock tea powder was added with 1 L of distilled water, sterilized at 121°C for 15 min, centrifuged, and the supernatant was taken) to make the moisture content of the rice slurry 55% to 60%. The rice slurry was inoculated with activated Bacillus coagulans PR06 (concentration: 2.0×10 7 cfu / mL, inoculation amount 2% v / v), ferment at 30-50°C for 8-18h, pour the fermented rice slurry into a self-cooking rice noodle extrusion machine, steam the obtained vermicelli for 2-10min, cool, age at 4-8°C for 3-10h, and finally dry to a moisture content of less than 14% to obtain a finished product. Figure 4 and Figure 5 A diagram showing the state of tea leaf rice vermicelli fermented by Bacillus coagulans PR06 before and after steaming.

[0044] Example 7 Detection of the cooking quality of tea vermicelli fermented by Bacillus coagulans PR06 The cooking quality of the rice vermicelli samples in Example 6 was detected, and the breaking rate, cooking loss rate and rehydration time of the rice vermicelli were determined with reference to the standard (SN / T 0395-2018), and the rehydration rate of the rice vermicelli was determined with reference to the standard (QB / T 2652-2004). The test results are shown in Table 4.

[0045] Table 4 The cooking quality test results of tea rice vermicelli fermented by Bacillus coagulans PR06

[0046] sample Strip breaking rate % Cooking loss% Rehydration rate % Rehydration time Embodiment 6 Fermented tea rice vermicelli 3.7±0.3 4.3±0.1 203.6±3.3 223±7 Control without PR06 inoculation 6.1±0.5 5.0±0.1 235.6±8.2 202±6

[0047] The results in Table 4 show that the fermented tea vermicelli prepared by using Bacillus coagulans PR06 of the present invention has good cooking quality, the breakage rate and cooking loss rate are both lower than 5.0%, the rehydration rate is greater than 200%, and the rehydration time is within 4 minutes (240 seconds).

[0048] Example 8 Detection of sensory quality of tea vermicelli fermented by Bacillus coagulans PR06

[0049] The sensory quality of the rice vermicelli in Example 6 was evaluated, and the results are shown in Table 5.

[0050] Table 5 Sensory evaluation criteria and results of tea rice vermicelli fermented by Bacillus coagulans PR06

[0051]

[0052]

[0053] The results in Table 5 show that the fermented tea rice vermicelli prepared by using the Bacillus coagulans PR06 of the present invention has excellent sensory quality, and the total sensory score reaches 92 points.

[0054] Example 9 Detection of flavor substances in tea vermicelli fermented by Bacillus coagulans PR06

[0055] The flavor substances in the fermented tea leaf rice vermicelli sample in Example 6 were detected.

[0056] Detection method: Take 2g of sample and transfer it into a 15mL extraction bottle, then quickly seal it. Aging the SPME extraction fiber head at 250℃ in the GC-MS injection port until there are no impurity peaks. Place the sample bottle on the solid phase microextraction device and set the temperature to 85℃; place the sample bottle in the extraction device and fully heat it in a water bath for 5min; insert the SPME extraction head into the headspace of the sample through the bottle cap, push out the fiber head, the extraction head is about 1.0cm above the upper surface of the sample, and the headspace is extracted for 45min; withdraw the fiber head and pull the extraction head out of the sample bottle; then insert the extraction head into the GC-MS injection port, push out the fiber head, analyze at 250℃ for 5min, and inject and analyze. GC detection conditions are as follows: chromatographic column: HP-5MS column (30m×0.25mm, 0.25μm); carrier gas: helium; flow rate 1mL / min; sample injection temperature maintained at 250℃; gas chromatograph oven temperature maintained at 40℃ for 3min, then increased to 150℃ at 4℃ / min, then increased to 250℃ at 10℃ / min, and then remained unchanged. MS conditions are as follows: EI ion source, electron energy 70eV, mass range 35-350AMU / s, GC-MS interface temperature 250℃, ion source temperature 230℃, quadrupole temperature 150℃.

[0057] The detection results of flavor substances in tea and rice vermicelli fermented by Bacillus coagulans PR06 are shown in Table 6.

[0058] Table 6 Detection results of flavor substances in tea rice vermicelli fermented by Bacillus coagulans PR06

[0059]

[0060]

[0061]

[0062] The results in Table 6 show that the main flavor substances of the fermented tea rice vermicelli prepared by using Bacillus coagulans PR06 of the present invention are nonanal, trans-2-heptanal, decanal, geraniol, tridecane, geranylacetone, 2,4-di-tert-butylphenol, octyl salicylate and diisobutyl phthalate.

[0063] In summary, the Bacillus coagulans PR06 provided by the present invention has strong acid and bile resistance, is resistant to most antibiotics, and can be used to prepare common fermented rice vermicelli or common fermented rice vermicelli strips, and can also be used to prepare fermented tea rice vermicelli or fermented tea rice vermicelli strips without bitterness and excellent in cooking and sensory quality. In view of the fact that Bacillus coagulans is one of the strains approved by my country for use in common food, and is a strain recognized in the industry as having high safety, the Bacillus coagulans PR06 provided by the present invention has high practical application value.

[0064] The above are only some specific embodiments of the present invention. The present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A strain of Bacillus coagulans ( Bacillus coagulans ) PR06, which features: It was deposited in the China Center for Type Culture Collection on December 10, 2021, with the deposit number: CCTCC NO: M 20211589, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The Bacillus coagulans according to claim 1 ( Bacillus coagulans ) Application of PR06 in the preparation of common fermented rice vermicelli or common fermented rice noodle products.

3. The Bacillus coagulans according to claim 1 ( Bacillus coagulans ) Application of PR06 in the preparation of fermented tea rice vermicelli or fermented tea rice vermicelli products.

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