CN116948865A

CN116948865A - Bacillus coagulans and application thereof

Info

Publication number: CN116948865A
Application number: CN202310473617.4A
Authority: CN
Inventors: 黄菊青; 林斌; 王�琦; 官雪芳
Original assignee: Institute of Agricultural Engineering Technology of Fujian Academy of Agricultural Sciences
Current assignee: Institute of Agricultural Engineering Technology of Fujian Academy of Agricultural Sciences
Priority date: 2023-04-28
Filing date: 2023-04-28
Publication date: 2023-10-27

Abstract

The application discloses a bacillus coagulans, which is classified and named as bacillus coagulansBacillus coagulans) PR06, which has been deposited at the China center for type culture Collection, under the accession number: cctccc NO: m20211589. The bacillus coagulans PR06 provided by the application has stronger acid resistance and cholate resistance, and has no resistance to most antibiotics. The application also discloses application of the bacillus coagulans in preparation of fermented rice vermicelli or fermented rice vermicelli products. The fermented tea rice vermicelli prepared by the bacillus coagulans PR06 provided by the application has no bitter and astringent taste, and is excellent in steaming and cooking and organoleptic quality and pleasant in flavor. Therefore, the bacillus coagulans PR06 provided by the applicationHas higher social application value.

Description

Bacillus coagulans and application thereof

Technical Field

The application relates to the technical field of microbial strains and application thereof, in particular to bacillus coagulans and application thereof.

Background

Bacillus coagulans (Bacillus coagulans) is a genus of the genus Geobacillus, the cell body is rod-shaped, the spore end grows, and no flagella exists, and is a facultative anaerobic gram positive bacterium. The bacillus coagulans can grow in aerobic and anaerobic environments, can adapt to low-oxygen intestinal environments, has higher tolerance to acid and bile, can perform lactic acid fermentation, can reduce the pH value of intestinal tracts, inhibit harmful bacteria, and can promote the growth and propagation of beneficial bacteria such as bifidobacteria. Bacillus coagulans is capable of sporulation and is beneficial for restoring microecological balance of the gastrointestinal tract as compared to other lactic acid-free bacilli. The bacillus coagulans spores can germinate in a human body for about 4-6 hours, wherein 85% of the thalli can smoothly pass through a digestive system and finally germinate and propagate in intestinal tracts. Compared with the intrinsic important lactobacillus and bifidobacterium in the gastrointestinal tract, the bacillus coagulans not only has the dual effects of the lactobacillus and the bifidobacterium, but also has the unique advantages of high temperature resistance, easy culture, stable storage and the like of the bacillus. Research shows that bacillus coagulans can improve the immunity of organisms, strengthen the disease resistance and promote the digestion and metabolism of the organisms and the absorption of nutrient substances. At present, bacillus coagulans has been listed by the department of agriculture in China as a novel feed additive which can be directly fed for livestock and poultry production. Bacillus coagulans is one of the approved species in China for use in common foods, and is announced by the national health and family planning Commission of the people's republic of China, 2014, no. 6. In the field of foods, bacillus coagulans is mainly applied to probiotics soft sweets and normal-temperature acidic drinks, and no report on application of bacillus coagulans to fermented tea rice vermicelli is currently seen in the industry.

Rice flour is a staple food common in southern areas of China, has hundreds of millions of eating population, and is a rice product with the largest market specification. In 2019, annual output values of Changde rice flour and Guilin rice flour in China respectively exceed 20 hundred million and 25 hundred million yuan. The rice flour can be classified into fermented rice flour and non-fermented rice flour, and researches show that the fermented rice flour has great improvement on taste and flavor, and has better steaming and boiling characteristics and rehydration. Because the common rice flour has single nutrient components (about 75% of starch and about 7% of protein), the common rice flour lacks health-care functional components. With the enhancement of health care consciousness of people in recent years, common rice flour can not meet the requirements of people on nutrition and health care. Tea is known as 'national drink' in China, and the health care effect of the tea is accepted by the masses, wherein the health care components mainly comprise tea polyphenol, theanine and the like. The tea food meets the expectations and the requirements of consumers on health care, and the application of the tea to various foods to be processed into foods with the characteristics of the tea and the health care effect is more and more popular among consumers. Because the starch in the rice flour is relatively easy to be digested and absorbed by human body, the blood sugar level of the human body is easy to be raised after eating, and the rice flour is not suitable for diabetics. Research shows that polyphenol substance can complex with starch macromolecules to change the crystal form of starch molecules, further influence the aging behavior and digestion property of starch gel, and play a role in resisting digestion. Therefore, tea powder containing polyphenol substances is added in the rice flour processing process, so that the effect of delaying digestion of rice flour can be achieved, and the rice flour is suitable for patients with hyperglycemia to eat. However, the rice flour may have a bitter taste due to tea polyphenols and caffeine in the tea leaves.

In order to solve the problems, the inventor aims at screening out strains for preparing the fermented tea rice vermicelli or rice noodles without bitter taste, and providing a new bacterial source for development and production of the fermented tea rice vermicelli or rice noodle products.

Disclosure of Invention

The application aims to provide bacillus coagulans and application thereof, and develops a strain which can be used for preparing fermented tea rice vermicelli (strip) without bitter taste.

The application aims at realizing the following technical scheme:

bacillus coagulans, designated Bacillus coagulans (Bacillus coagulans) PR06, deposited on the China center for type culture Collection, accession number: cctccc NO: m20211589, accession number address: eight 299 channels of Wuchang district of Wuhan, hubei province, china center for type culture Collection, post code: 430072.

the bacillus coagulans PR06 provided by the application is separated from human excrement and has the colony characteristics that: after being cultured on an MRS flat plate culture medium for 24-48 hours, the bacterial colony is in a milky white, round, smooth in surface, moist and glossy shape; the scanning electron microscope observation shows that the bacterial cells are rod-shaped, and the size is 0.2-0.4x2.0-5.0 mu m; the optimum growth temperature is 50 ℃.

The application also provides application of the bacillus coagulans PR06 in processing and producing common fermented rice vermicelli or common fermented rice noodles.

The application also provides application of the bacillus coagulans PR06 in processing and producing fermented tea rice vermicelli or fermented tea rice noodles.

The application has the remarkable advantages that:

the bacillus coagulans PR06 provided by the application has stronger acid resistance and cholate resistance, and has no resistance to most antibiotics. The common fermented rice vermicelli prepared by the bacillus coagulans PR06 provided by the application has the advantages of excellent organoleptic quality, unique flavor and pleasure. The fermented tea rice vermicelli prepared by the bacillus coagulans PR06 provided by the application has no bitter and astringent taste, and is excellent in steaming and cooking and organoleptic quality and pleasant in flavor. Therefore, the bacillus coagulans PR06 provided by the application has higher social application value.

Drawings

Fig. 1 is a colony morphology of bacillus coagulans PR06.

Fig. 2 is a scanning electron microscope image of bacillus coagulans PR06.

Fig. 3 is a graph showing the growth of bacillus coagulans PR06 at different temperatures.

Fig. 4 is a diagram of a finished product of the bacillus coagulans PR06 fermented tea leaf and rice flour noodles (before cooking).

Fig. 5 is a diagram of a finished product of the bacillus coagulans PR06 fermented tea leaf and rice flour noodles (after cooking).

Detailed Description

The following examples facilitate a better understanding of the present application, but are not intended to limit the same. The experimental methods in the following examples are conventional media and experimental conditions unless otherwise specified. The test materials, reagents and the like used in the examples are all commercially available unless otherwise specified.

EXAMPLE 1 isolation and characterization of Bacillus coagulans (Bacillus coagulans) PR06

1. Isolation of strains

Taking 1.0g of fresh human excrement sample, placing the sample into a sterile centrifuge tube filled with 9.0mL of sterile water, shaking and uniformly mixing, placing the obtained bacterial suspension into boiling water at 80 ℃ for water bath for 30min, and taking out; sequentially diluting with 10-fold gradient dilution method, selecting 100 μL of diluent with proper concentration gradient, adding MRS agar (tryptone 10.0g/L, beef powder 5.0g/L, yeast powder 4.0g/L, glucose 20.0g/L, tween 80 1.0mL/L, K) ₂ HPO ₄ ·3H ₂ O1.5 g/L, anhydrous sodium acetate 3.0g/L, ammonium citrate 2.0g/L, mgSO ₄ ·7H ₂ O 0.2g/L、MnSO ₄ ·H ₂ O0.04 g/L, agar powder 20.0g/L, distilled water 1L and pH value of 6.2-6.4), uniformly coating by using a sterile coating rod, taking sterile water as a blank control, and placing the coated sterile water in a constant temperature incubator at 45 ℃ for inversion culture for 48-72 h. The white or off-white, glossy, round, clean-edged, single-colony strain was picked, and repeatedly streaked, purified and cultured on MRS agar plate medium until a single colony was obtained, and the single-colony strain was designated PR06.

2. Identification of strains

(1) Morphology observation of colonies and thalli: strain PR06 was streaked on MRS agar plate medium and incubated at 37 ℃ for 24-48 hours, and the colony morphology (fig. 1) showed milky white, round, smooth surface, moist and glossy. The bacterial cells are in the shape of rod and the size is 0.2-0.4 μm×2.0-5.0 μm when observed under a scanning electron microscope, and the scanning electron microscope diagram is shown in FIG. 2.

(2) Growth characteristics observation of strain PR 06: the strain PR06 bacterial suspension is respectively inoculated into MRS liquid culture medium according to the inoculum size of 3.0 percent of the volume ratio, and is cultured for 24 hours at different temperatures, and the strain grows well in the environment of 30-60 ℃, and the optimal growth temperature is 50 ℃ (see figure 3).

(3) Molecular biological identification of strain PR 06: the whole genome of the strain PR06 is extracted by using a bacterial whole genome rapid extraction kit, and PCR is performed by selecting bacterial 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') and then sequencing and analyzing. The sequencing result is compared by BLAST, the strain is identified as bacillus coagulans, the gene sequence is shown in SEQ ID NO.1, and the sequence is named as: bacillus coagulans (Bacillus coagulans) PR06 deposited at the China center for type culture Collection, 12:10, 2021, accession number: cctccc NO: m20211589. Deposit unit address: eight 299 channels of Wuchang district of Wuhan, hubei province, china center for type culture Collection, post code: 430072.

example 2 availability of bacillus coagulans (Bacillus coagulans) PR06 to saccharides API50CHL kit (Biomerieux, france) the strain PR06 isolated in example 1 was tested for availability to saccharides and the results (table 1) show: bacillus coagulans (Bacillus coagulans) PR06 of the present application can utilize ribose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetylglucosamine, esculin ferric citrate, D-maltose, D-lactose, D-melibiose, D-sucrose, trehalose, D-raffinose and starch.

Table 1 results of testing the availability of sugars by strain PR06 using API50CHL kit

Substrate composition	48h reaction results	Substrate composition	48h reaction results
				Control	-	Esculin ferric citrate	+
Glycerol	-	Salicin (Liu Chun)	-
				Erythritol	-	D-cellobiose	-
D-arabinose	-	D-maltose	+
				L-arabinose	-	D-lactose	+
Ribose	+	D-melibiose	+
				D-xylose	-	D-sucrose	+
L-xylose	-	Trehalose	+
				Adonitol	-	Inulin (inulin)	-
beta-methyl-D-xylosides	-	D-melezitose	-
				D-galactose	+	D-raffinose	+
D-glucose	+	Starch	+
				D-fructose	+	Glycogen	-
D-mannose	+	Xylitol	-
				L-sorbose	-	D-gentiobiose (in Niu Ertang)	-
L-rhamnose	-	D-turbinase (D-melezitose)	-
				Celastrol	-	D-lyxose	-
Inositol (inositol)	-	D-tagatose	-
				Mannitol (mannitol)	+	D-fucose (D-fucose)	-
Sorbitol	+	L-fucose	-
				alpha-methyl-D-mannopyranoside	-	D-arabitol (D-arabitol)	-
alpha-methyl-D-glucopyranoside	-	L-arabitol (L-arabitol)	-
				N-acetylglucosamine	+	Potassium gluconate (salt)	-
Amygdalin	-	2 Keto-gluconic acid potassium salt	-
				ARBULIN (arbutin)	-	5 Keto-gluconic acid potassium salt	-

Remarks: + represents a positive result, indicating that the strain is able to utilize the test substrate; -a negative result indicating that the strain is unable to utilize the test substrate.

EXAMPLE 3 acid and bile salt resistance of Bacillus coagulans (Bacillus coagulans) PR06

Preparing activated strain PR06 into suspension, and collecting 1.0mL of the bacterial suspension (concentration is 2.0X10) ⁷ cfu/mL) was inoculated into 9.0mL of MRS liquid medium having pH of 2.5, 3.0, 3.5 or bile salt concentration of 1.0, 2.0, 3.0 and 4.0g/L, respectively, cultured at 45℃for 4 hours, viable count was measured by plate count method at 0 hours after the culture and 4 hours after the culture, respectively, and survival rate (%) was calculated.

Survival rate (%) = (number of viable bacteria for 4 h/number of viable bacteria for 0 h) ×100.

TABLE 2 acid and bile salt resistance of Bacillus coagulans PR06

The results in Table 2 show that Bacillus coagulans PR06 has higher survival rate under the environments of pH of 3.0, 3.5 or bile salt concentration of 1.0, 2.0, 3.0 and 4.0g/L, which indicates that the strain has stronger acid resistance and bile salt resistance and can smoothly pass through the gastrointestinal tract to enter the large intestine of the organism to play a probiotic role.

EXAMPLE 4 antibiotic resistance of Bacillus coagulans (Bacillus coagulans) PR06 broth was inoculated into MRS broth, cultured in a 45℃biochemical incubator for 24 hours, 100. Mu.L broth was spread on MRS agar plates, and tablets containing erythromycin, penicillin, gentamicin, kanamycin, streptomycin, bacitracin, chloramphenicol, vancomycin, tetracycline, ampicillin, cefoxitin, rifampin, neomycin, nalidixic acid, ciprofloxacin, polymyxin were placed thereon, and cultured upside down in a 45℃biochemical incubator for 24 hours, and the size of the inhibition zone was observed and recorded. The experimental results are shown in table 3.

TABLE 3 bacteriostatic Effect of antibiotics on Bacillus coagulans PR06

The results in Table 3 show that Bacillus coagulans PR06 was only resistant to both bacitracin and polymyxin, but not to most other antibiotics, indicating that PR06 had good safety for use.

Example 5 preparation and sensory evaluation of Bacillus coagulans PR06 ordinary fermented rice vermicelli the early long-grained nonglutinous rice with an amylose content of more than 20% and aged for 1 to 1.5 years was milled and sieved through a 80-mesh sieve to obtain rice flour, 800g of the rice flour sample was taken, 150g of corn starch and 50g of potato starch were added, and after thoroughly mixing, the mixture was spread under an ultraviolet lamp and sterilized for 24 hours, and a proper amount of pure water was added to make the moisture content of the rice slurry 55 to 60%. Inoculating activated Bacillus coagulans PR06 (concentration: 2.0X10) on ultra-clean bench ⁷ cfu/mL, inoculum size 2% v/v), fermenting at 30-50deg.C for 8-18 h, and fermentingPouring the fermented rice pulp into a self-cooked silk-extrusion molding rice flour machine, steaming the obtained vermicelli for 2-10 min, cooling, aging for 3-10 h at 4-8 ℃, and finally drying until the water content is lower than 14%, thus obtaining the finished product. Sensory evaluation of the finished vermicelli shows that the bacillus coagulans PR06 common fermented rice vermicelli has good sensory quality and pleasant sweet and sour feeling when chewed.

Example 6 preparation of Bacillus coagulans PR06 fermentation type tea Rice vermicelli

Grinding long-shaped rice which has the amylose content of more than 20% and is aged for 1-1.5 years, sieving the long-shaped rice with a 80-mesh sieve to obtain rice flour, taking 800g of rice flour sample, adding 150g of corn starch and 50g of potato starch, fully and uniformly mixing, spreading the mixture under an ultraviolet lamp for sterilization for 24 hours, adding a proper amount of tea water (taking 50g of rock tea powder, adding 1L of distilled water, sterilizing for 15 minutes at 121 ℃, centrifuging, taking supernatant) and enabling the moisture content of rice pulp to be 55% -60%. Inoculating activated Bacillus coagulans PR06 (concentration: 2.0X10) on ultra-clean bench ⁷ cfu/mL, inoculum size 2% v/v), fermenting at 30-50 ℃ for 8-18 h, pouring the fermented rice milk into a self-cooked silk extrusion molding rice flour machine, steaming the obtained vermicelli for 2-10 min, cooling, aging at 4-8 ℃ for 3-10 h, and finally drying until the water content is lower than 14%, thus obtaining the finished product. Fig. 4 and 5 show the state diagrams before and after steaming of the bacillus coagulans PR06 fermented tea-leaf rice noodles.

Example 7 detection of steaming quality of tea leaf rice vermicelli fermented by bacillus coagulans PR06 the steaming quality of the rice vermicelli sample in example 6 was detected, specifically the breakage rate, steaming loss rate and rehydration time of the rice vermicelli were determined with reference to the standard (SN/T0395-2018), and the rehydration rate of the rice vermicelli was determined with reference to the standard (QB/T2652-2004). The measurement results are shown in Table 4.

TABLE 4 measurement of cooking quality of Bacillus coagulans PR06 fermented tea Rice vermicelli

Sample of	The breakage rate is%	Cooking loss rate%	Rehydration percentage%	Rehydration time s
					Example 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

The results in Table 4 show that the fermented tea rice noodles prepared by using the bacillus coagulans PR06 of the application have good steaming quality, the breakage rate and the steaming loss rate are both lower than 5.0%, the rehydration rate is higher than 200%, and the rehydration time is within 4 minutes (240 s).

Example 8 detection of sensory quality of Bacillus coagulans PR06 fermented tea Rice vermicelli

The sensory quality of the rice vermicelli in example 6 was evaluated, and the results are shown in table 5.

TABLE 5 sensory evaluation criteria and results for Bacillus coagulans PR06 fermented tea and rice vermicelli

The results in Table 5 show that the fermented tea rice noodles prepared by using the Bacillus coagulans PR06 of the present application are excellent in sensory quality, and the total sensory score reaches 92 minutes.

Example 9 detection of flavour substances in Bacillus coagulans PR06 fermented tea Rice vermicelli

The flavor substances in the fermented tea rice noodle sample in example 6 were detected.

The detection method comprises the following steps: a2 g sample was removed into a 15mL extraction flask and then quickly sealed. The SPME extracted fiber heads were aged at 250deg.C to no impurity peaks at the GC-MS sample inlet. Placing the sample bottle on a solid-phase microextraction device, and setting the temperature to be 85 ℃; placing the sample bottle in an extraction device, and fully heating in water bath for 5min; inserting the SPME extraction head into the head space of the sample through the bottle cap, pushing out the fiber head, wherein the extraction head is about 1.0cm higher than the upper surface of the sample, and performing head space extraction for 45min; the fiber head is withdrawn, and the extraction head is pulled out of the sample bottle; and then the extraction head is inserted into a GC-MS sample inlet, the fiber head is pushed out, analysis is carried out for 5min at 250 ℃, and sample injection analysis is carried out. The GC detection conditions were as follows: chromatographic column: HP-5MS column (30 m.times.0.25 mm,0.25 μm); carrier gas: helium gas; the flow rate is 1mL/min; the sample injection temperature was maintained at 250 ℃; the gas chromatograph oven temperature was maintained at 40 ℃ for 3min, then increased to 150 ℃ at 4 ℃/min, then to 250 ℃ at 10 ℃/min, and then kept unchanged. The MS conditions were as follows: EI ion source, electron energy 70eV, mass range 35-350 AMU/s, GC-MS interface temperature 250 ℃, ion source temperature 230 ℃, quadrupole rod temperature 150 ℃.

The detection results of the flavor substances of the fermented tea rice vermicelli by the bacillus coagulans PR06 are shown in table 6.

TABLE 6 detection results of flavor substances of Bacillus coagulans PR06 fermented tea Rice vermicelli

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The results in Table 6 show that the main flavors of the fermented tea rice vermicelli prepared by using Bacillus coagulans PR06 of the application are nonanal, trans-2-heptanal, decanal, geraniol, tridecanol, geranylacetone, 2, 4-di-tert-butylphenol, octyl salicylate, diisobutyl phthalate and the like.

In conclusion, the bacillus coagulans PR06 provided by the application has strong acid resistance and bile salt resistance, has resistance to most antibiotics, can be used for preparing common fermented rice vermicelli or common fermented rice noodles, and can also be used for preparing fermented tea rice vermicelli or fermented tea rice noodles which have no bitter taste and good steaming and sensory quality. In view of the fact that bacillus coagulans is one of the approved strains for common foods in China and belongs to the strains which are accepted in the industry and have high safety, the bacillus coagulans PR06 provided by the application has high practical application value.

The foregoing is only a few specific embodiments of the present application. The application is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present application.

Claims

1. A bacillus coagulans strain, characterized in that: the strain is classified and named as bacillus coagulansBacillus coagulans) PR06, which has been deposited at the China center for type culture Collection, under the accession number: cctccc NO: m20211589, the preservation address is: no. 299 is eight paths in Wuchang district of Wuhan, hubei province.

2. The use of bacillus coagulans according to claim 1 for the preparation of a common fermented rice vermicelli or a common fermented rice noodle product.

3. The use of bacillus coagulans according to claim 1 for the preparation of fermented tea rice vermicelli or fermented tea rice vermicelli products.