CN109706098B - Lactobacillus paracasei and application thereof - Google Patents
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Abstract
The invention discloses a Lactobacillus paracasei (Lactobacillus paracasei), and the preservation number of the Lactobacillus paracasei is CGMCC No. 16200. The invention also discloses a fermented product and a preparation method thereof, and application of the Lactobacillus paracasei (Lactobacillus paracasei) in preparation of the fermented product. Compared with the existing strains, the lactobacillus paracasei CGMCC No.16200 provided by the invention can rapidly ferment soybean protein, shortens the initial coagulation time, improves the texture, taste, flavor (beany flavor) and the like of the prepared fermented soybean milk, still maintains higher viable count after being stored for 27 days at 4 ℃, and has good gastrointestinal environment tolerance.
Description
Technical Field
The invention relates to the field of microorganisms, and particularly relates to lactobacillus paracasei and application thereof.
Background
Soybean, which is ancient called "Shu", originated from China, is rich in nutrients and is an ideal plant protein resource. The amino acid composition of the soybean protein is similar to that of milk protein, and the amino acid is necessary to be rich except for slightly low methionine content; and the soybean protein is the only food crop which is close to full-valence protein in the plant protein and is also called as 'green milk'. The soybean milk is a traditional neutral soybean protein milk, contains rich high-quality protein, has the proportion of amino acid close to that required by a human body, and is beneficial to the absorption of the human body. In addition, the soybean milk also contains a large amount of active ingredients, such as soybean phospholipids, which have the function of removing blood vessel garbage and are called as 'scavenger' of blood vessels, and can promote the growth and development of cells; the soybean isoflavone has effects of estrogen-like and bidirectional hormone level regulation; selenium, vitamin C, vitamin E and the like have strong antioxidant function and certain functions of delaying senility and resisting cancer.
The fermented soybean milk is prepared by fermenting soybean milk with lactobacillus to produce acid, so as to coagulate soybean protein. The fermented product not only has the nutrient components in the soybean milk, but also increases the quantity of intestinal probiotics, vitamins and other substances, has the functions of promoting intestinal peristalsis, promoting digestion, enhancing immunity and the like, and ensures that the soybean milk has more nutrient and health-care effects. Compared with fermented milk, the fermented sour soybean milk does not contain cholesterol, contains a large amount of polyunsaturated fatty acid, and has very wide market development prospect in preventing heart diseases, diabetes, cardiovascular diseases, osteoporosis, lactose intolerance people and the like.
At present, most researches on soybean milk are limited to products such as soybean milk powder and liquid soymilk, or fermented products obtained by adding auxiliary materials such as milk and skim milk powder, and the researches on preparing fermented soybean milk by using pure soybeans as a single fermentation raw material are a main direction of future researches.
Disclosure of Invention
The invention aims to improve the tolerance of a fermentation strain for preparing fermented soybean milk in gastrointestinal tracts, improve the texture characteristics, taste and flavor (beany flavor) of the fermented soybean milk, stabilize the storage period of the fermented soybean milk and shorten the milk clotting time, thereby providing a lactobacillus paracasei and application thereof in preparing the fermented soybean milk.
In order to achieve the above object, in a first aspect, the present invention provides a Lactobacillus paracasei (Lactobacillus paracasei) having a collection number of CGMCC number 16200.
In a second aspect, the present invention provides a fermentation inoculum comprising Lactobacillus paracasei and Lactobacillus salivarius CGMCC No.16199 as described above.
In a third aspect, the present invention provides the use of a lactobacillus paracasei as described above or a fermentation broth as described above for the preparation of fermented soy milk.
In a fourth aspect, the present invention provides a fermented soybean milk containing the lactobacillus paracasei as described above or the fermentation inoculant as described above.
In a fifth aspect, the present invention provides a method for preparing fermented soybean milk, comprising: the lactobacillus paracasei or the zymogen agent is inoculated to the soybean milk to be fermented and fermented.
The invention can obtain the following beneficial effects:
(1) the lactobacillus paracasei CGMCC No.16200 (hereinafter referred to as lactobacillus paracasei M11-4) can rapidly ferment soybean protein, the initial coagulation time is 2.8 hours, the time is obviously shorter than that of other existing strains, and the effect is better when the lactobacillus paracasei CGMCC No.16199 is combined for use.
(2) The sensory evaluation scores of the texture, the taste, the flavor (beany flavor) and the like of the fermented soybean milk prepared by the lactobacillus paracasei M11-4 provided by the invention are all obviously better than those of other existing strains, and the effect is better when the fermented soybean milk is combined with the lactobacillus salivarius CGMCC No. 16199.
(3) The lactobacillus paracasei M11-4 provided by the invention has good stability in a fermented soybean milk system, still keeps higher viable count when stored for 27 days at 4 ℃, is higher than other existing strains, and has better effect when used in combination with lactobacillus salivarius CGMCC No. 16199.
(4) The lactobacillus paracasei M11-4 provided by the invention has good gastrointestinal tract environment tolerance, and can still maintain 10 after being treated for 4 hours in the environment with the pH of 2.07.2CFU/mL viable count; the product can be processed in artificial gastric juice environment for 4 hr and still maintain 10%8.9The viable count of CFU/mL is 10 after being treated for 4 hours in the environment of artificial gastric juice9.1The viable count of CFU/mL is obviously higher than that of other existing strains.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Biological preservation
Lactobacillus paracasei
The Lactobacillus paracasei (Lactobacillus paracasei) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: No. 3 of Xilu 1 of Beijing City facing Yang district, Ministry of microbiology, China institute of microbiology, zip code: 100101) in 2018, 8 and 6 months (the preservation unit is abbreviated as CGMCC), and the preservation number is CGMCC No.16200, which is abbreviated as M11-4.
Lactobacillus salivarius
The Lactobacillus salivarius is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: No. 3 of West Lu No.1 of Beijing and Kogyo area, North Chen, China academy of sciences, Microbiol research institute, postal code: 100101) 8.8.8.6.2018.8.6.6 (the preservation unit is abbreviated as CGMCC), and the preservation number is CGMCC No.16119, which is abbreviated as M18-6.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
In one aspect, the invention provides Lactobacillus paracasei (Lactobacillus paracasei), wherein the preservation number of the Lactobacillus paracasei is CGMCC No. 16200.
The lactobacillus paracasei is separated from the natural fermented rice flour of Guangxi Bama village.
The Lactobacillus paracasei provided by the present invention can produce a large amount of viable cells of Lactobacillus paracasei by liquid culture, and the culture method is not particularly limited as long as the Lactobacillus paracasei can proliferate, and may be, for example, 107Inoculating the living strain of the lactobacillus paracasei into a lactobacillus culture medium at the inoculation amount of CFU/mL, and culturing at the temperature of 15-38 ℃ for 8-72 hours under the anaerobic or aerobic condition to obtain a culture solution. The above-mentionedThe culture medium for Lactobacillus may be any medium suitable for culturing Lactobacillus known in the art, for example, milk and/or Lactobacillus (MRS) medium as described in "Lactobacillus-biological basis and applications" (Poplar, light industry Press, 1996).
In the present invention, the viable cells of lactobacillus paracasei in the culture solution may be further separated, and the method for separating is not particularly limited as long as the cells can be enriched from the culture solution, and for example, the separation may be performed by centrifugation and/or filtration, and the conditions for centrifugation and filtration may be known conditions, and the present invention is not described herein again.
In a second aspect, the present invention provides a fermentation inoculant comprising Lactobacillus paracasei and Lactobacillus salivarius CGMCC No.16199 as described above.
The lactobacillus salivarius is separated from natural fermented rice flour of Guangxi Bama village.
The Lactobacillus salivarius provided by the present invention can produce a large amount of viable cells of Lactobacillus salivarius by liquid culture, and the culture method is not particularly required as long as it can proliferate the Lactobacillus salivarius, and may be, for example, 107Inoculating the live lactobacillus salivarius in a lactobacillus culture medium at the inoculation amount of CFU/mL, and culturing at 15-38 ℃ for 8-72 hours under anaerobic or aerobic conditions to obtain a culture solution. The culture medium for the Lactobacillus may be any medium suitable for culturing Lactobacillus known in the art, such as milk and/or Lactobacillus (MRS) medium described in "Lactobacillus-biological basis and applications" (Poplar, light industry Press, 1996).
In the present invention, the viable cells of lactobacillus salivarius in the culture solution can be further separated, and the method for separating is not particularly limited as long as the cells can be enriched from the culture solution, and for example, the separation can be achieved by a method of centrifugation and/or filtration, and the conditions of centrifugation and filtration can be known conditions, and the present invention is not described herein again.
According to the invention, the fermentation microbial inoculum can be prepared by carrying out fermentation culture on the lactobacillus paracasei and the lactobacillus salivarius in a fermentation culture medium, wherein the two strains can be subjected to mixed culture or can be subjected to mixing after independent culture. The fermentation medium may be any of various media conventionally used in the art for fermenting lactic acid bacteria, for example, MRS medium as described above, and may be any of various media suitable for fermenting lactic acid bacteria optimized based on MRS medium.
According to the present invention, the fermentation conditions may be conventional conditions for lactic acid bacteria fermentation culture well known in the art, for example, the temperature of the fermentation culture may be 30-40 ℃.
According to the invention, the preparation method of the zymophyte agent can be prepared according to the conventional method in the field, and in a preferred embodiment of the invention, the zymophyte agent can be prepared according to the following method: culturing Lactobacillus paracasei CGMCC No.16200 until the viable count reaches 108More than cfu/mL, culturing Lactobacillus salivarius CGMCC No.16119 until viable count reaches 108More than cfu/mL, uniformly mixing, and then carrying out vacuum freeze-drying to obtain the product.
In a third aspect, the present invention provides the use of a lactobacillus paracasei as described above or a fermentation broth as described above for the preparation of fermented soy milk.
In a fourth aspect, the invention also provides fermented soybean milk containing the lactobacillus paracasei or the zymophyte agent as described above.
In a fifth aspect, the present invention provides a method for preparing fermented soybean milk, comprising: the lactobacillus paracasei as described above or the fermentation inoculum as described above is inoculated into the soybean milk to be fermented and fermented.
According to the invention, the lactobacillus paracasei, or the lactobacillus paracasei and the lactobacillus salivarius, inoculated into the soybean milk to be fermented are live bacteria. However, it is known to those skilled in the art that the cell death is usually accompanied in the process of cell culture, and the living cells are not all living cells in a strict sense.
The invention is a pair for inoculationThe number of cells of Lactobacillus casei and Lactobacillus salivarius is not particularly limited, and can be specifically adjusted depending on the intended use. Preferably, the lactobacillus paracasei inoculation amount is 10 in terms of the lactobacillus paracasei on the basis of each milliliter or each gram of raw material to be fermented6-108CFU, the inoculation amount of Lactobacillus salivarius is 106-108CFU。
CFU (Colony-Forming Units) refers to the number of viable bacteria. In the case of viable bacteria culture counting, a colony formed by growing and propagating a single cell or a plurality of aggregated cells on a solid medium is called a colony forming unit, and the number of viable bacteria is expressed.
According to the invention, in order to further improve the fermentation performance, the soybean milk preferably also contains white granulated sugar, and the content of the white granulated sugar is preferably 4-6g based on 100g of the soybean milk.
According to the present invention, in order to further improve the flavor and mouthfeel of the finally prepared fermented soybean milk, it is preferable that the method further comprises, in the end of the fermentation, cold-preserving the fermented material at 2 to 6 ℃ for 10 to 20 hours for after-ripening.
According to the invention, the fermentation conditions may be carried out according to conditions conventional in the art, for example, the temperature of the fermentation may be between 30 ℃ and 40 ℃, wherein the time of fermentation is based on the formation of clots. The lactobacillus paracasei provided by the invention can be used for fermenting soybean milk, so that the fermentation time can be obviously shortened, and particularly when the lactobacillus paracasei and the lactobacillus salivarius are used in combination.
According to the present invention, the soybean milk to be fermented may be soybean milk.
The following preparation examples, examples and comparative examples will further illustrate the present invention, but do not limit the present invention accordingly.
In the following preparations, examples and comparative examples:
experimental strains: the lactobacillus paracasei of the invention (the strain is preserved in the common microorganism center of China microorganism culture preservation management committee (address: No. 3 of West Lu No.1 of Beijing city, Chaoyang, North Chen province, and the microbiological research institute of Chinese academy of sciences, zip code: 100101) (the preservation unit is abbreviated as CGMCC), the preservation number is CGMCC No.16200) in 8 and 6 days of 2018, and is hereinafter referred to as a strain M11-4;
the Lactobacillus salivarius is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: No. 3 of Xilu 1 of Beijing & Kogyo, No. 3 of the national academy of sciences, China institute of microbiology, postal code: 100101) (the abbreviation of preservation Unit is CGMCC) in 8.8.8.6.2018, and the preservation number is CGMCC No.16119, M18-6 for short
The strains for comparison are Lactobacillus plantarum Zhang-ll (CGMCC No.6936, CN201410763406.5), Lactobacillus casei KL1(CGMCC No.1809), Lactobacillus bulgaricus (Lactobacillus bulgaricus) (CGMCC No.1809)
11842D-5) and Streptococcus thermophilus (S.thermophilus)
BAA-491)。
MRS liquid medium formula: 2g of dimethyl hydrogen phosphate, 5g of anhydrous sodium acetate, 5g of yeast extract powder, 0.5g of magnesium sulfate heptahydrate, 10g of beef extract, 2g of ammonium citrate, 20g of glucose, 10g of tryptone, 0.25g of manganese sulfate, 801 mL of tween, 1000mL of distilled water (20 g of agar added in a solid culture medium), pH6.5, and sterilization at 115 ℃ for 15min for later use, wherein the reagents are purchased from Corbai biotechnology company;
the titration acidity determination method comprises the following steps: 10g of fermented soybean milk, 20mL of distilled water and 2mL of 0.1 vol% phenolphthalein alcohol solution are added, the mixture is shaken up and titrated to red color by using 0.1moL/L NaOH standard solution, and the color can not fade within 30 s. Acidity ° T is measured as milliliters of 0.1M NaOH consumed for 100mL of sample.
The viable cell count measuring method comprises the following steps: adding 1ml of fermented milk into a 9ml of 0.85% physiological saline tube, sequentially carrying out gradient dilution by 10 times, selecting 3 appropriate dilution gradients, adding 1ml of diluent into a 90mm culture dish, pouring and adding 15ml of MRS solid culture medium at 55 ℃, slightly mixing uniformly, standing, solidifying, inverting, placing into a 37 ℃ constant temperature incubator, and counting the total number of colonies after culturing for 48 hours.
Preparation example
This preparation example is intended to illustrate the activation of the strains
The strains of lactobacillus paracasei M11-4, lactobacillus salivarius M18-6, lactobacillus plantarum Zhang-ll, lactobacillus casei KL1, lactobacillus bulgaricus and streptococcus thermophilus glycerol tube preserved which are frozen in an ultra-low temperature refrigerator at minus 80 ℃, are respectively inoculated into an MRS liquid culture medium according to the inoculum concentration of 2 volume percent, then are cultured for 12 hours under the conditions of 37 ℃ and 180r/min, and are continuously activated for 3 generations for subsequent tests.
Examples
This example serves to illustrate the fermentation performance of the strains provided by the invention
(1) Mixing soybean milk (commercially available from Tianrunfeng agriculture science and technology development (Beijing) Co., Ltd.) and white sugar at a weight ratio of 100:5, and sterilizing at 115 deg.C for 15min to obtain soybean milk fermented base material.
(2) Respectively 10 in the clean bench7CFU/mL total inoculum size respectively prepares activated lactobacillus paracasei M11-4, lactobacillus salivarius M18-6, lactobacillus plantarum Zhang-ll, lactobacillus casei KL1, lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus casei M11-4+ lactobacillus salivarius M18-6 (inoculum size 1: 1), lactobacillus casei M18-6+ lactobacillus plantarum Zhang-ll (inoculum size 1: 1), inoculates the activated lactobacillus paracasei M11-4, lactobacillus casei M11-4+ lactobacillus salivarius M18-6 and lactobacillus casei Zhang-ll of the examples into the soybean milk fermentation base material, fully oscillates and uniformly mixes, then ferments in a constant temperature incubator at 37 ℃, judges that the fermentation end point arrives after the soybean milk is primarily solidified, stops fermenting, records the fermentation time, and results are shown in Table 1.
(3) And (3) rapidly cooling the fermented material to 4 ℃, and refrigerating at the low temperature of 4 ℃ for 12 hours to obtain the fermented soybean milk.
TABLE 1 curd time for different strains of fermented soymilk
As can be seen from the table 1, the lactobacillus paracasei CGMCC No.16200 provided by the invention can rapidly ferment soybean protein, the initial coagulation time is 2.8 hours, is obviously shorter than that of other existing strains, and the effect is better by combining with lactobacillus salivarius M18-6.
Test example
(1) Evaluation of storage stability of fermented Soybean milk
The fermented soybean milks (Lactobacillus paracasei M11-4, Lactobacillus salivarius M18-6, Lactobacillus plantarum Zhang-ll, Lactobacillus casei KL1, Lactobacillus casei M11-4+ Lactobacillus salivarius M18-6) prepared in examples were stored at 4 ℃ respectively, changes in pH, acidity and viable cell count every 3 days were measured, and the results of continuous measurement for 27 days were shown in tables 2 to 4, where Table 2 shows changes in pH, Table 3 shows changes in acidity, and Table 4 shows changes in viable cell count.
TABLE 2 pH change during storage of fermented sour soymilk with different strains
TABLE 3 variation of acidity (0T) of fermented sour soymilk with different strains during storage
TABLE 4 variation of viable count (lg CFU/mL) of fermented sour milk of different strains in storage period
As can be seen from tables 2-4, the lactobacillus paracasei M11-4 provided by the invention has good stability (less change) in a fermented soybean milk system, still keeps higher viable count after being stored for 27 days at 4 ℃, has better performance than other existing strains, and has better effect by combining with lactobacillus salivarius M18-6.
(2) Sensory evaluation of fermented soymilk
10 professionally trained sensory evaluators (aged between 20 and 26 years) were selected and evaluated in accordance with the following Table 5, and the evaluation results are shown in Table 6.
TABLE 5 sensory evaluation criteria for sour soymilk
TABLE 6 sensory evaluation of fermented soymilk
| Fermentation strain | Color | Taste of the product | Smell(s) | Tissue state |
| Lactobacillus paracasei M11-4 | 30 | 58 | 57 | 52 |
| Lactobacillus salivarius M18-6 | 30 | 60 | 58 | 53 |
| Lactobacillus plantarum Zhang-ll | 30 | 56 | 55 | 50 |
| Lactobacillus casei KL1 | 30 | 52 | 50 | 48 |
| Lactobacillus casei M11-4+ Lactobacillus salivarius M18-6 | 30 | 60 | 60 | 55 |
As can be seen from Table 6, the fermented soybean milk prepared from Lactobacillus paracasei M11-4 provided by the invention has the advantages of significantly improved texture, mouthfeel, beany flavor and other properties compared with other strains, and better effect when combined with Lactobacillus salivarius M18-6.
(3) Gastrointestinal environmental tolerance
1. Evaluation of acid resistance of Strain
And centrifuging the activated 2-generation bacterial liquid (lactobacillus paracasei M11-4, lactobacillus salivarius M18-6 and lactobacillus plantarum Zhang-ll) at 4000r/min for 10min, and washing the obtained precipitate twice by using normal saline. The pellet was resuspended in MRS medium (pH 2.0) and cultured at 37 ℃ and 180r/min for 4 hours, and the viable cell count was determined, and the results are shown in Table 7.
2. Evaluation of strains in simulated gastrointestinal tolerance
The activated bacteria liquid is centrifuged (4000r/min, 10min) to obtain a precipitate, the precipitate is suspended in artificial gastric juice and intestinal juice, the artificial gastric juice and the intestinal juice are stressed for 4h, and the survival rate of the thallus after stress is calculated, and the result is shown in table 7.
TABLE 7 viable count of different strains after 4 hours under simulated gastrointestinal conditions
| Fermentation strain | Acid resistance | Artificial gastric juice | Artificial intestinal juice |
| Lactobacillus paracasei M11-4 | 7.22 | 8.86 | 9.06 |
| Lactobacillus salivarius M18-6 | 8.58 | 8.77 | 9.22 |
| Lactobacillus plantarum Zhang-ll | 6.00 | 6.02 | 6.74 |
As can be seen from Table 7, the Lactobacillus paracasei M11-4 provided by the invention has good gastrointestinal tract environment tolerance, and can still maintain 10 after being treated for 4 hours in the environment with the pH of 2.07.2CFU/mL viable count; the product can be processed in artificial gastric juice environment for 4 hr and still maintain 10%8.9The viable count of CFU/mL is 10 after being treated for 4 hours in the environment of artificial gastric juice9.1The viable count of CFU/mL is obviously improved compared with other strains.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the respective technical features described in the above embodiments may be combined in any suitable manner without contradiction. The invention is not described in detail for the various possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (9)
1. A Lactobacillus paracasei (Lactobacillus paracasei) is characterized in that the preservation number of the Lactobacillus paracasei is CGMCC No. 16200.
2. A fermentation inoculum comprising Lactobacillus paracasei and Lactobacillus salivarius CGMCC No.16199 according to claim 1.
3. Use of the lactobacillus paracasei of claim 1 or the fermenting agent of claim 2 for preparing fermented soybean milk.
4. A fermented soybean milk characterized by containing the Lactobacillus paracasei of claim 1 or the fermentation inoculant of claim 2.
5. A method for preparing fermented soybean milk, which is characterized by comprising the following steps: inoculating the lactobacillus paracasei of claim 1 or the fermentation inoculum of claim 2 into soybean milk to be fermented, and fermenting.
6. The method according to claim 5, wherein the lactobacillus paracasei inoculum size in terms of lactobacillus paracasei is 10 per ml of soy milk6-108CFU, the inoculation amount of Lactobacillus salivarius is 106-108CFU。
7. The method according to claim 5 or 6, wherein the soy milk further comprises white granulated sugar, the content of white granulated sugar being 4-6g based on 100g of the soy milk.
8. The method of any of claims 5-7, wherein the method further comprises: and (3) during the fermentation, refrigerating the fermented material for 10-20 hours at the temperature of 2-6 ℃.
9. The process according to any one of claims 5 to 7, wherein the temperature of the fermentation is 30-40 ℃.
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