CN113355374A - Method for synthesizing B vitamins by fermenting lactobacillus paracasei ET-22 and application - Google Patents

Method for synthesizing B vitamins by fermenting lactobacillus paracasei ET-22 and application Download PDF

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CN113355374A
CN113355374A CN202011348863.XA CN202011348863A CN113355374A CN 113355374 A CN113355374 A CN 113355374A CN 202011348863 A CN202011348863 A CN 202011348863A CN 113355374 A CN113355374 A CN 113355374A
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vitamin
fermentation
vitamins
lactobacillus paracasei
milk
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CN113355374B (en
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洪维鍊
赵雯
刘伟贤
刘福东
侯保朝
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Inner Mongolia Yili Industrial Group Co Ltd
Inner Mongolia Dairy Technology Research Institute Co Ltd
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Inner Mongolia Yili Industrial Group Co Ltd
Inner Mongolia Dairy Technology Research Institute Co Ltd
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Abstract

The invention provides a method for synthesizing B vitamins by fermenting lactobacillus paracasei ET-22 and application thereof. The invention firstly provides a method for synthesizing B vitamins by fermenting Lactobacillus paracasei (Lactobacillus paracasei) ET-22, which comprises the following steps: the fermentation product containing B vitamins is prepared by fermenting lactobacillus paracasei CGMCC No. 15077. The research of the invention finds that the lactobacillus paracasei strain can be used for the production and application of fermented milk, milk drinks, daily chemicals, pet food, probiotics for animal husbandry and the like which are rich in B vitamins.

Description

Method for synthesizing B vitamins by fermenting lactobacillus paracasei ET-22 and application
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for synthesizing B vitamins by fermenting Lactobacillus paracasei ET-22 (preservation number CGMCC No.15077) and related application.
Background
The B vitamins are also called B vitamins (also called vitamin B, vitamin B group or vitamin B complex group), and are classified as "families" because of 8 types in total. The B vitamins are water-soluble vitamins which are indispensable for maintaining the normal functions and metabolic activities of human bodies, and can be quickly consumed in the human bodies in the tense life and working pressure, improper dietary habits or due to the use of certain specific medicines and the property of dissolving the B vitamins in water. The supplement of vitamin B can relieve emotion. Moreover, according to research reports, the B vitamins can help to maintain the functions of the heart and the nervous system, maintain the health of the digestive system and the skin, participate in energy metabolism, enhance physical strength, tonify and strengthen the body. If vitamin B is absent, cell function is immediately reduced, and metabolic disorder is caused, at which the human body suffers from malaise and inappetence. Furthermore, excessive drinking and the like cause liver damage, which is concurrent with vitamin B deficiency in many cases.
The B vitamins are widely present in rice bran, yeast, animal liver, coarse grain vegetables and other foods. Studies have shown that part of the B vitamins can be synthesized in the human gut.
Lactobacillus paracasei is a gram-positive facultative heterotrophic lactic acid bacterium commonly used in dairy fermentation and probiotics. Lactobacillus paracasei is commonly found in a variety of human habitats, such as the human intestinal tract and oral cavity, as well as silage and naturally fermented foods.
No relevant research report about the B vitamins produced by the fermentation of lactobacillus paracasei is found.
Disclosure of Invention
One object of the present invention is to provide a novel use of Lactobacillus paracasei ET-22.
Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain has been preserved in China general microbiological culture Collection center (CGMCC) at 18 days 12 months in 2017 (address: Beijing, West Lu No.1 Hospital No. 3 of the rising district, China academy of sciences), and is classified and named: lactobacillus paracasei (Lactobacillus paracasei); the preservation number is CGMCC No. 15077.
The invention discovers that Lactobacillus paracasei (Lactobacillus paracasei) ET-22 takes feed liquid (milk substrate) mainly containing milk components as a fermentation substrate, and has high-yield B vitamins (containing vitamin B)1Vitamin B2Vitamin C and vitamin CElement B3Vitamin B6Vitamin B9) The ability of the cell to perform.
Thus, in one aspect, the present invention provides a method for the fermentative synthesis of B vitamins by Lactobacillus paracasei (Lactobacillus paracasei), comprising: the fermentation product containing B vitamins is prepared by fermenting lactobacillus paracasei CGMCC No. 15077.
According to a particular embodiment of the invention, the method of the invention further comprises: extracting and purifying B vitamins from the supernatant of the fermentation product.
According to a specific embodiment of the present invention, the B vitamins include vitamin B1Vitamin B2Vitamin B3Vitamin B6And vitamin B9One or more of (a).
According to a specific embodiment of the present invention, the B vitamins include vitamin B3And/or vitamin B9。
According to a particular embodiment of the invention, the fermentation substrate used in the fermentation may be an emulsion. Preferably, the milk may be raw milk or reconstituted milk, and may be whole milk, low fat milk or skim milk. The emulsion may optionally contain 5% to 10% sucrose.
According to a specific embodiment of the present invention, the fermentation conditions are: fermenting for 1-7 days at 35-45 ℃.
According to a specific embodiment of the invention, the lactobacillus paracasei is inoculated in the fermentation substrate in the form of seed liquid, with an inoculation amount of 2% to 5%. The preparation of the seed liquid may be carried out according to conventional techniques in the field of probiotics.
According to a specific embodiment of the present invention, the milk substrate used as the fermentation substrate of lactobacillus paracasei ET-22 in the present invention may be milk (whole milk, low-fat milk or skim milk) with milk content of 80% or more, wherein auxiliary materials such as sweeteners and stabilizers may be optionally added according to need, for example, fermentation substrate commonly used in the preparation of fermented milk or milk beverage, and the present invention may be applied to the milk substrateThe lactobacillus paracasei ET-22 can realize the effect of high yield of B vitamins by fermenting the milk substrate, and the fermentation product is particularly rich in vitamin B1Vitamin B2Vitamin B3Vitamin B6Vitamin B9。
Vitamin B1The content in milk is 0.3 mg/L on average. Vitamin B in milk1The lactobacillus fermented sour milk is not completely from feed, and can be synthesized by microorganisms in rumen of cow, so that vitamin B in sour milk fermented by lactobacillus is more than that in common milk1The content is increased by about 30 percent. Children less than one week in age need vitamin B daily15-10 mg. If vitamin B is deficient1It can cause anorexia, slow growth, damage to nervous system, polyneuritis, milk regurgitation or constipation, dyspepsia, green stool, pale complexion, inappetence, muscle relaxation, frequent crying, and weight loss in children. According to some embodiments of the invention, the vitamin B in the Lactobacillus paracasei ET-22 fermented emulsion1The content can reach 60-80 mu g/mL. The yogurt product is administered in an amount of 100 mL/day and is supplemented with VB for the recommended human intake1。
Vitamin B2In human milk, the content of vitamin B in cow milk is not high2The content can reach 1-2 mg/L. The children are recommended to take 21-3 mg vitamin B every day. If vitamin B2 is lacked, it will cause growth retardation, anorexia, inflammation of mouth horn and lip, red and swollen tongue, malnutrition, and asthenia. According to some embodiments of the invention, the vitamin B in the Lactobacillus paracasei ET-22 fermented emulsion2The content can reach 2.8-3 mu g/mL.
Vitamin B3(nicotinic acid) is recommended to be taken by adults at 13-19mg per day (dietary guidelines of Chinese residents), 20mg per day is recommended for pregnant women, and 22mg per day is recommended for pregnant women in lactation. Vitamin B3It is mainly used in livestock meat, fish and poultry, and plant food such as mushroom, asparagus, sunflower seed, peanut, bean and grain. Vitamin B in ordinary yoghurt3Is present in an amount of about 0.2mg/100 g.According to some embodiments of the invention, the vitamin B in the Lactobacillus paracasei ET-22 fermented emulsion3Is 50-80 mu g/mL and is far higher than the content of the common yoghourt.
Vitamin B6Also known as pyridoxine, which includes pyridoxine, pyridoxal and pyridoxamine, exists in vivo in the form of phosphate esters, and is a water-soluble vitamin. Vitamin B6The functionality involves a number of aspects including: participating in protein synthesis and catabolism and participating in all amino acid metabolism; participating in gluconeogenesis and UFA metabolism, and is related to glycogen metabolism, sphingomyelin metabolism and steroid metabolism; participate in the synthesis of certain nerve mediators (5-hydroxytryptamine, taurine, dopamine, norepinephrine and gamma-aminobutyric acid); involving nucleic acid and DNA synthesis, lacking vitamin B6DNA synthesis is impaired, a process which is important for maintaining proper immune function. Papastoyianidis et al (2006) use commercial ferments YO-1, YO-2 to ferment at 42 ℃ for 3.5h, then store for 1.5h in cold storage, detect vitamin B6The content of (A) is 1.37mg/kg and 1.50mg/kg respectively; vitamin B after fermentation assisted by Bifidobacterium BB-12 and Lactobacillus acidophilus LA-56The contents of (A) were 1.37mg/kg and 1.31mg/kg, respectively. According to some embodiments of the invention, the vitamin B in the Lactobacillus paracasei ET-22 fermented emulsion6The content is 7-14 mug/mL.
Vitamin B9The content of the (folic acid) in the cow milk is generally 40 +/-10.00 mu g/L, and the content of the (folic acid) in the common yoghurt is 80 +/-20.00 mu g/L. Vitamin B9The main effects on organisms are as follows: are involved in the metabolism of genetic material and proteins; influence animal reproductive performance; affecting secretion of the animal pancreas; promoting the growth of animals; improving immunity of organism. Vitamin B9As one of the essential vitamins for the growth and reproduction of body cells, the deficiency can affect the normal physiological activities of human bodies. Many reports in the literature of vitamin B deficiency9Has direct relation with diseases such as neural tube deformity, megaloblastic anemia, cleft lip and palate, depression, tumor and the like. In general, normal persons require vitamin B9The amount of the compound is 100-200 mu g/d, and the recommended amount of the world health organization is as follows: adult 200 ug/d, pregnant womanAnd cow breast 400. mu.g/d. The latest folic acid consumption standard of the FDA is as follows: between 25 and 50 years old (male) 240 μ g/d; 190 μ g/d between 25 and 50 years old (female); the concentration of the extract is 400 mug/d for the nannie and the pregnant woman; the infant is 200-400 mu g/d. According to some embodiments of the invention, the vitamin B in the Lactobacillus paracasei ET-22 fermented emulsion9The content of (b) is 2.5-2.9 [ mu ] g/mL.
Thus, in another aspect, the invention also provides the use of lactobacillus paracasei in the preparation of a composition rich in B vitamins by fermentation, wherein the fermentation product rich in B vitamins is prepared by fermentation of lactobacillus paracasei CGMCC No. 15077.
According to a particular embodiment of the invention, the lactobacillus paracasei of the invention is used in the fermentative preparation of a composition enriched in B vitamins, which may be a food composition, a daily chemical composition or a pharmaceutical composition. Specifically, the fermentation product containing B vitamins obtained by the fermentation of the present invention can be used as such or further used for preparing foods (e.g., fermented milk, milk drinks, milk powder, solid beverages, etc.), daily chemicals (cosmetics, etc.), feeds (including pet foods or probiotic products for livestock), or medicines. The method for preparing the food, daily chemical, feed or pharmaceutical from the fermentation product can be carried out with reference to the prior art in the field.
In a particular embodiment of the invention, the B vitamin containing fermentation product of the invention is used as or further for the preparation of a food product, preferably a fermented milk or fermented milk beverage. The emulsion used as a fermentation substrate may further comprise other materials commonly used in fermented milk or fermented milk beverages, such as appropriate amounts of sweeteners, stabilizers, nutritional additives, and the like.
According to a particular embodiment of the invention, the fermented milk matrix of lactobacillus paracasei of the invention results in a fermentation product containing B vitamins which is also enriched in conjugated linoleic acid and/or gamma-aminobutyric acid. Therefore, the invention also provides the application of the lactobacillus paracasei ET-22 in the preparation of a composition rich in conjugated linoleic acid and/or gamma-aminobutyric acid through fermentation. In the composition, conjugated linoleic acid and/or gamma-aminobutyric acid are mainly produced by fermentation of the lactobacillus paracasei.
In conclusion, the invention provides the new application of lactobacillus paracasei ET-22, and the invention discovers that the single-strain fermented milk substrate of the strain has good vitamin B inclusion synthesized by vitamin B1Vitamin B2Vitamin B3Vitamin B6Vitamin B9The ability of the cell to perform. The lactobacillus paracasei ET-22 strain can be used for producing fermented milk, milk drinks, daily chemicals and the like rich in B vitamins.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description taken in conjunction with the accompanying specific embodiments, and the technical solutions of the present invention are described, it being understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Example 1: production of B vitamins from Lactobacillus paracasei fermented milk
1. Experimental strains
The lactobacillus paracasei ET-22(CGMCC No.15077) used in the technical scheme of the invention;
control strain 1: lactobacillus paracasei strain l.casei 431;
control strain 2: the commercial strain Lactobacillus paracasei LPC-37.
2. Activation of strains and liquid culture of seeds
(1) Strain activation: activating lactobacillus paracasei by using MRS culture medium, taking the strain growing to logarithmic phase, and preserving glycerol tubes for later use. Wherein, MRS culture medium (g/L): peptone 10.0 g; 5.0g of yeast extract; 20.0g of glucose; 5.0g of anhydrous sodium acetate; 2.0g of dihydroamine citrate; tween-801.0 mL; dipotassium phosphate 2.0 g; magnesium sulfate heptahydrate 0.2 g; 0.05g of manganese sulfate; 10.0g of beef extract; 15g to 20g of agar.
(2) Determination of growth dynamics of strains
Dissolving 12% whole milk powder and 6% white sugar in water at normal temperature, sterilizing at 95 deg.C for 5min, cooling to 36 + -1 deg.C, inoculating strain as milk matrix, fermenting and culturing for 120h, sampling at different fermentation time periods, and measuring viable count and pH value.
And (3) viable count detection: after mixing 25ml of the sample with 75ml of physiological saline uniformly, counting by a plate dilution method.
And (3) acidity detection: the sample pH was measured using a pH meter and recorded. Taking 10g of the fermented dairy product, adding 2 drops of phenolphthalein solution, mixing uniformly, and titrating 0.1M standard NaOH solution to pink. The calculation formula of the titration acidity is as follows:
°T＝C×V×100/(m×0.1)。
the results are shown in tables 1 and 2, respectively.
TABLE 1 colony count of Lactobacillus paracasei in milk matrix for different fermentation times
Figure BDA0002800802010000051
Figure BDA0002800802010000061
According to the analysis of the test data, the ET-22 has the advantages that the viable count is increased rapidly at 28h, and then the decrease is stable. The viable count of the control strain 1 and the control strain 2 is increased rapidly after 36 hours, and then the viable count is reduced to be stable. ET-22 is significantly different from control strain 1 and control strain 2 (p is less than 0.05); control strain 1 and control strain 2 analyzed p >0.05 with no significant difference. Wherein the blank group is an emulsion matrix without added bacteria liquid.
TABLE 2 pH determination of Lactobacillus paracasei in milk substrates for different fermentation times
Figure BDA0002800802010000062
Figure BDA0002800802010000071
Analysis of the test data shows that the pH value of ET-22 fermentation is gradually reduced within 28-72 h, and the pH values of the control strain 1 and the control strain 2 are gradually reduced within 12-36 h. The ET-22 has extremely obvious difference with p <0.01 analyzed by a control strain 1 and a control strain 2; control strain 1 and control strain 2 analyzed p >0.05 with no significant difference. The presence of non-killed micro-organisms in the blank resulted in milk spoilage and a decrease in pH due to temperature. Wherein the blank group is an emulsion matrix without added bacteria liquid.
(3) Preparation of strain seed liquid: dissolving 12% whole milk powder and 6% white sugar in water at normal temperature, sterilizing at 95 deg.C for 5min, cooling to 36 + -1 deg.C, inoculating strain, fermenting at 36 + -1 deg.C for 48 + -2 h, and prolonging to 72 + -2 h to obtain seed liquid.
3. Synthesis of B vitamins from lactobacillus paracasei fermented milk
Preparing fermented milk: adding sucrose accounting for 6.5 percent of the total weight of the raw milk into the raw milk, stirring, heating to 95 ℃, sterilizing for 5min, cooling the sterilized emulsion to 37 ℃, respectively adding various bacterial seed liquids (the inoculum size is 3 percent), fermenting the samples at 37 ℃ for 24, 48, 72, 96 and 120h respectively, centrifuging the fermented emulsion at 8000r/min for 15min, collecting the fermented supernatant, filtering the fermented supernatant through a 0.22 mu m filter membrane, and measuring the content of the B vitamins in the filtrate.
The content of B vitamins is determined by high performance liquid chromatography, and the specific operation conditions refer to HPLC detection of water-soluble vitamins (Liyan, food research and development, 2018) in Collybia nigricans.
Blank milk base (without added probiotics) was used as a control, and blank control supernatant could not be obtained by centrifugation due to the inability to curd during the same fermentation time. Therefore, the following data are the vitamin contents directly measured in the supernatant of the fermentation broth, without deducting the vitamin contents in the blank milk matrix.
(1) Vitamin B in fermentation samples1In an amount of
Vitamin B in probiotic fermentation supernatant with different fermentation times1See table 3 for the contents of (d).
TABLE 3 fermentation supernatant vitamin B1In an amount of
Figure BDA0002800802010000072
Figure BDA0002800802010000081
From the analysis of the above test data, it can be seen that vitamin B is present in the fermentation supernatant of each strain1The content showed a clear rising trend with time. Differences were found between ET-22, control strain 1, and control strain 2, but were not significant.
(2) Fermentation sample vitamin B2In an amount of
Vitamin B in probiotic fermentation supernatant with different fermentation times2See table 4 for the contents of (d).
TABLE 4 fermentation supernatant vitamin B2In an amount of
Figure BDA0002800802010000082
From the analysis of the above test data, it can be seen that vitamin B in the supernatant of the fermentation of each strain was observed at different times2The contents are all close and the difference is not obvious. Wherein vitamin B in the supernatant in the first 72h2The content shows a clear ascending trend, and the content is basically unchanged after 48 h.
(3) Fermentation sample vitamin B3In an amount of
Vitamin B in probiotic fermentation supernatant with different fermentation times3See table 5 for the contents of (d).
TABLE 5 vitamin B in fermentation supernatants3In an amount of
Figure BDA0002800802010000083
From the analysis of the above test data, it can be seen that vitamin B is present in the fermentation supernatant of each strain3The content of the strain is obviously increased along with the time, and the control strain 2 is increasedThe potential is relatively slow. Vitamin B in supernatant of ET-22 and control Strain 1 when fermented for 120h3The content difference is obvious (p is less than 0.05). Vitamin B in supernatant after 72h fermentation of ET-22 and control Strain 23The content difference is obvious (p is less than 0.05).
(4) Fermentation sample vitamin B6In an amount of
Vitamin B in probiotic fermentation supernatant with different fermentation times6See table 6 for the contents of (d).
TABLE 6 vitamin B in fermentation supernatants6In an amount of
Figure BDA0002800802010000091
From the analysis of the above test data, it can be seen that vitamin B is present in the fermentation supernatant of each strain6The content showed a clear rising trend with time, the rising trend of the control strain 2 was relatively slow. Vitamin B in supernatant after 120h fermentation of ET-22 and control Strain 16The content was varied but not significant.
(5) Fermentation sample vitamin B9In an amount of
Vitamin B in probiotic fermentation supernatant with different fermentation times9See table 7 for the contents of (d).
TABLE 7 vitamin B9In an amount of
Figure BDA0002800802010000092
From the analysis of the test data, the vitamin B in the supernatant of the ET-22120 h fermentation9The content was about 1.5 times that of the control strain 1. Vitamin B in fermentation supernatant of each strain9The content of vitamin B is substantially constant with time9The contents were all at a lower level. ET-22 and control Strain 1, control Strain 2 vitamin B in fermentation supernatants9The content difference is very obvious (P is less than 0.01).
According to the experimental result of the viable count of each strain, the lactobacillus paracasei reaches the maximum value of the viable count within about 48 hours, namely the stable period, and the viable counts are kept unchanged after 48 hours. After 24h fermentation, fermentation supernatant can not be obtained to detect the content of folic acid, but folic acid is supposed to be metabolized and generated in the process of strain growth. There was no significant difference in the folate content over time during the subsequent fermentation time, a possible reason: the folic acid content reaches the maximum value in the stable growth period (48h) of the strain, and as the strain is continuously metabolized, the thalli age and the folic acid synthesis amount is reduced; the influence of environmental factors such as light, heat, temperature and the like on the folic acid content in the experimental process.
(6) Content of Conjugated Linoleic Acid (CLA) and gamma-aminobutyric acid in fermentation sample
In the invention, beneficial substances such as Conjugated Linoleic Acid (CLA) and gamma-aminobutyric acid are generated in the ET-22 fermented milk. See tables 8 and 9 for relevant test results. Wherein, the content determination method of the conjugated linoleic acid is based on the gas chromatography NY/T1671-2008 for the content determination of the Conjugated Linoleic Acid (CLA) in milk and dairy products; the method for measuring the content of the gamma-aminobutyric acid is based on QB/T4587-2013 gamma-aminobutyric acid.
TABLE 8 content of conjugated linoleic acid in the ET-22 fermentation supernatant
Figure BDA0002800802010000101
TABLE 9 content of gamma-aminobutyric acid in the supernatant from ET-22 fermentation
Figure BDA0002800802010000102
Example 2 fermentation of Lactobacillus paracasei ET-22 to prepare a vitamin B enriched milk drink
Heating fresh milk to more than 50 ℃, adding 6% of white granulated sugar, stirring until the white granulated sugar is completely dissolved, preheating to 60-65 ℃, homogenizing under the pressure of 20Mpa, carrying out heat treatment for 5-8 minutes at about 90 ℃, cooling to 37 ℃, and inoculating lactobacillus paracasei ET-22 seed liquid, wherein the inoculation amount is 2-3%. Fermenting at 37 deg.C for 96-120 h. Diluting the fermented product with sterilized and cooled mixture of water, syrup, emulsion stabilizer, etc. 2-4 times to obtain beverageThe lactobacillus beverage is used. The lactobacillus beverage has sour and sweet taste, good flavor, and high vitamin B content3Vitamin B9Conjugated linoleic acid and gamma-aminobutyric acid content.

Claims (10)

1. A method for synthesizing B vitamins by fermenting Lactobacillus paracasei (Lactobacillus paracasei), which comprises the following steps: the fermentation product containing B vitamins is prepared by fermenting lactobacillus paracasei CGMCC No. 15077.
2. The method of claim 1, further comprising: extracting and purifying B vitamins from the supernatant of the fermentation product.
3. The method according to claim 1 or 2, wherein the B vitamins comprise vitamin B1Vitamin B2Vitamin B3Vitamin B6And vitamin B9One or more of (a).
4. The method of claim 3 wherein the B vitamins comprise vitamin B3And/or vitamin B9。
5. The method according to claim 1, wherein the fermentation substrate used in the fermentation is an emulsion; preferably, the emulsion is fresh milk or reconstituted milk, and optionally contains 5-10% of sucrose.
6. The method of claim 1 or 5, wherein the fermentation conditions are: fermenting for 1-7 days at 35-45 ℃.
7. The method according to claim 1 or 5, wherein the Lactobacillus paracasei is inoculated in the fermentation substrate in the form of seed liquid, in an amount of 2% to 5%.
8. The application of lactobacillus paracasei in preparing the composition rich in B vitamins by fermentation, wherein the fermentation product rich in B vitamins is prepared by fermenting lactobacillus paracasei with CGMCC No. 15077; the obtained fermentation product containing B vitamins can be used as or further used for preparing food (such as fermented milk, milk beverage, milk powder, solid beverage, etc.), daily chemical (cosmetics, etc.), feed (including pet food or probiotic product for animal husbandry) or medicine.
9. Use according to claim 8, wherein the food product is a fermented milk or a fermented milk beverage.
10. Use according to claim 8 or 9, wherein the composition is also enriched in conjugated linoleic acid and/or gamma-aminobutyric acid, produced by fermentation of said lactobacillus paracasei.
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