CN116240139B - Lactobacillus delbrueckii subspecies bulgaricus LB02, bacterial powder and bacterial strain combination for producing conjugated linoleic acid and application thereof - Google Patents
Lactobacillus delbrueckii subspecies bulgaricus LB02, bacterial powder and bacterial strain combination for producing conjugated linoleic acid and application thereof Download PDFInfo
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
Abstract
The invention relates to Lactobacillus delbrueckii subspecies bulgaricus LB02 for producing conjugated linoleic acid, bacterial powder, bacterial strain combination and application thereof, wherein the Lactobacillus delbrueckii subspecies bulgaricus LB02 is named as Lactobacillus delbrueckii subspecies bulgaricusLactobacillus delbrueckii subsp bulgaricus LB02, deposited in China general microbiological culture Collection center with the preservation number of CGMCC No.24664, the preservation date of 2022, 4 months and 11 days, and the preservation address of North Chen Xili No.1, 3 in the Chaoyang area of Beijing city. The Lactobacillus delbrueckii subsp bulgaricus LB02 can convert linoleic acid into conjugated linoleic acid, has high conversion rate, and can be used in industrial production of bioconversion of conjugated linoleic acid.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and relates to lactobacillus delbrueckii subspecies bulgaricus LB02, bacterial powder and bacterial strain combination for producing conjugated linoleic acid and application thereof.
Background
Conjugated Linoleic Acid (Conjugated Linoleic Acids, CLA) is a generic term for the position and geometric isomer of Linoleic Acid (LA) with conjugated double bonds, which is one of the indispensable fatty acids of functional unsaturated fatty acids, human and animals, but is a substance which cannot be synthesized by itself to have remarkable pharmacological action and nutritional value.
Conjugated linoleic acid is a novel nutrient, is favored in the health food industry at present, and is beneficial to human health. A large number of documents prove that the conjugated linoleic acid has certain free radical scavenging capability, can resist oxidation and tumors, effectively regulate blood viscosity, resist atherosclerosis, improve immunity, improve bone density, promote growth, prevent and treat physiological functions such as diabetes. In recent years, some clinical researches report that conjugated linoleic acid plays a role in reducing fat by inhibiting the activity of lipoprotein in fat cells, preventing degradation of apolipoprotein, promoting fat transportation, inhibiting synthesis of fat. The research shows that the human body intakes 3g/d conjugated linoleic acid has positive effect on preventing cancer, but because the conjugated linoleic acid content obtained from traditional food is less, more and more foods are exogenously added with conjugated linoleic acid, so that the conjugated linoleic acid has a certain health care function.
The conjugated linoleic acid synthesis method is divided into chemical synthesis and biological synthesis, and the conjugated linoleic acid synthesized by the chemical method has complex components, various isomer forms and poor safety, and is not suitable for being added into food or medical use. The principle of biosynthesis is that some microorganisms produce linoleic acid isomerase in cells, which converts linoleic acid into conjugated linoleic acid. Recent studies have shown that linoleic acid isomerase can be derived from a variety of microorganisms, such as LIN et al (Food chemistry.1999 (67) 1-5) by adding 1mg/mL linoleic acid to skim milk medium, inoculating Lactobacillus acidophilusLactobacillus acidophilus) Fermenting for 48h, and extracting and detecting to obtain conjugated sub-componentOleic acid yield was 0.052-0.094mg/mL; zhang Zhongyi (Industrial microorganism 2004.34 (2) 5-9) separating and screening from Chinese sauerkraut juice to obtain Lactobacillus plantarum strain with high conjugated linoleic acid production capabilityLactobacillus plantarum) The micro-aerobic condition can improve the yield of conjugated linoleic acid. CN1772911A discloses a strain of Lactobacillus plantarumLactobacillus plantarumHSC 235) CCTCC No. m204051d has the capacity of isomerizing linoleic acid structure to generate conjugated linoleic acid in the culture solution and buffer solution; the method for producing the active milk beverage rich in conjugated linoleic acid by biological transformation of lactobacillus plantarum is disclosed in CN102144667B, and the biological enrichment of conjugated linoleic acid by lactobacillus casei CGMCC1.574 is disclosed in CN 104404092B.
At present, the invention of lactobacillus bulgaricus (namely lactobacillus delbrueckii bulgaricus) capable of converting conjugated linoleic acid is not disclosed, and the yield of conjugated linoleic acid in most reported researches is low, so that a novel strain with high conversion rate of conjugated linoleic acid is important for pushing the industrial production industry of conjugated linoleic acid and developing related functional foods and medicines.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide lactobacillus delbrueckii subspecies bulgaricus LB02, bacterial powder, bacterial strain combination for producing conjugated linoleic acid and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a conjugated linoleic acid producing Lactobacillus delbrueckii subspecies bulgaricus LB02, said Lactobacillus delbrueckii subspecies bulgaricus LB02 being designated Lactobacillus delbrueckii subspecies bulgaricusLactobacillus delbrueckii subsp bulgaricusLB02, deposited in China general microbiological culture Collection center with the preservation number of CGMCC No.24664, the preservation date of 2022, 4 months and 11 days, and the preservation address of North Chen Xili No.1, 3 in the Chaoyang area of Beijing city.
In a second aspect, the invention provides lactobacillus delbrueckii subsp bulgaricus LB02 powder, wherein the lactobacillus delbrueckii subsp bulgaricus LB02 powder comprises lactobacillus delbrueckii subsp bulgaricus LB02 producing conjugated linoleic acid and a protective agent.
Preferably, the protective agent comprises any one or a combination of at least two of trehalose, maltodextrin, sucrose or tween-80, for example, the combination of trehalose and maltodextrin, the combination of sucrose and tween-80, the combination of trehalose and sucrose, and the like, and any other combination mode can be adopted.
Preferably, the protective agent comprises 15-35 parts by weight of trehalose, 1-15 parts by weight of maltodextrin, 1-5 parts by weight of sucrose and 0.01-0.5 part by weight of tween-80.
Specific values in the 15 to 35 parts are, for example, 15 parts, 17 parts, 20 parts, 22 parts, 25 parts, 27 parts, 30 parts, 32 parts, 35 parts, and the like.
Specific values in the above 1 to 15 parts are, for example, 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, and the like.
Specific values in the above 1 to 5 parts are, for example, 1 part, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, etc.
Specific values among the above 0.01 to 0.5 parts are, for example, 0.01 part, 0.03 part, 0.05 part, 0.07 part, 0.1 part, 0.15 part, 0.2 part, 0.25 part, 0.3 part, 0.35 part, 0.4 part, 0.45 part, 0.5 part, and the like.
In a third aspect, the invention provides a preparation method of lactobacillus delbrueckii subspecies bulgaricus LB02 powder, which is characterized in that lactobacillus delbrueckii subspecies bulgaricus LB02 is cultivated to obtain a culture solution, the culture solution is centrifuged, bacterial sludge is collected, mixed with a protective agent and freeze-dried to obtain the lactobacillus delbrueckii subspecies bulgaricus LB 02.
Preferably, the temperature of the culture is 32-37 ℃, e.g., 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃,37 ℃ and the like, and the time of the culture is 10-20 hours, e.g., 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours and the like.
Preferably, the cultivation is performed at a pH of 6.0-6.8, e.g., 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, etc.
Preferably, the culture medium used in the culture contains glucoseGlucose, lactose, peptone, beef extract, yeast extract, K 2 HPO 4 Diammonium hydrogen citrate, tween-80, sodium acetate and CaCl 2 、MgSO 4 Or MnSO 4 Any one or a combination of at least two of these.
Preferably, the components in the culture medium comprise 20-40g/L glucose, 5-10g/L lactose, 10-30g/L peptone, 3-10g/L beef extract powder, 3-10g/L, K yeast extract powder 2 HPO 4 2-5g/L, 2-5g/L diammonium hydrogen citrate, 2-5g/L sodium acetate, and 0.5-1.0g/L, mgSO Tween-80 4 0.1-0.5g/L、CaCl 2 0.1-0.5g/L and MnSO 4 0.05-0.1g/L。
Specific values among the above 20 to 40g/L are, for example, 20g/L, 22g/L, 24g/L, 26g/L, 28g/L, 30g/L, 32g/L, 34g/L, 36g/L, 38g/L, 40g/L, etc.
Specific values among the above 5 to 10g/L are, for example, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, etc.
Specific values among the above 10 to 30g/L are, for example, 10g/L, 12g/L, 14g/L, 16g/L, 18g/L, 20g/L, 22g/L, 24g/L, 26g/L, 28g/L, 30g/L, etc.
Specific values among the above 3 to 10g/L are, for example, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, etc.
Specific values among the above 2 to 5g/L are, for example, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5g/L, etc.
Specific values among the above 0.5 to 1.0g/L are, for example, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1.0g/L, etc.
Specific values among the above 0.1 to 0.5g/L are, for example, 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, etc.
Specific values among the above 0.05 to 0.1g/L are, for example, 0.05g/L, 0.06g/L, 0.07g/L, 0.08g/L, 0.09g/L, 0.1g/L, etc.
Preferably, the method further comprises activating the strain before culturing, specifically inoculating the strain into a seed culture medium for activating culture;
preferably, the temperature of the activation culture is 32-37 ℃, for example 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃,37 ℃ and the like.
Preferably, the seed culture medium contains glucose, lactose, peptone, beef extract, yeast extract, K 2 HPO 4 Diammonium hydrogen citrate, tween-80 and MgSO 4 And MnSO 4 。
Preferably, the components in the seed culture medium comprise 10-20g/L glucose, 10-15g/L lactose, 5-10g/L peptone, 5-15g/L beef extract powder, 2-6g/L, K 2 HPO 4 2-3g/L, diammonium hydrogen citrate 1-3g/L, tween-80 0.5-1.0g/L, mgSO 4 0.3-0.5g/L and MnSO 4 0.05-0.1g/L。
Specific values among the above 10 to 20g/L are, for example, 10g/L, 12g/L, 14g/L, 16g/L, 18g/L, 20g/L, etc.
Specific values among the above 10 to 15g/L are, for example, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, etc.
Specific values among the above 5 to 10g/L are, for example, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, etc.
Specific values among the above 5 to 15g/L are, for example, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, etc.
Specific values among the above 2 to 6g/L are, for example, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5g/L, 5.5g/L, 6g/L, etc.
Specific values among the above 2 to 3g/L are, for example, 2g/L, 2.1g/L, 2.2g/L, 2.3g/L, 2.4g/L, 2.5g/L, 2.6g/L, 2.7g/L, 2.8g/L, 2.9g/L, 3g/L, etc.
Specific values among the above 1 to 3g/L are, for example, 1g/L, 1.2g/L, 1.5g/L, 1.7g/L, 2g/L, 2.2g/L, 2.5g/L, 2.7g/L, 3g/L, etc.
Specific values among the above 0.5 to 1.0g/L are, for example, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1.0g/L, etc.
Specific values among the above 0.3 to 0.5g/L are, for example, 0.3g/L, 0.32g/L, 0.35g/L, 0.37g/L, 0.4g/L, 0.42g/L, 0.45g/L, 0.47g/L, 0.5g/L, etc.
Specific values among the above 0.05 to 0.1g/L are, for example, 0.05g/L, 0.06g/L, 0.07g/L, 0.08g/L, 0.09g/L, 0.1g/L, etc.
Preferably, the rotational speed of the centrifugation is 4000-7000r/min, e.g. 4000r/min, 4500r/min, 5000r/min, 5500r/min, 6000r/min, 6500r/min, 7000r/min etc. and the time of the centrifugation is 10-15min, e.g. 10min, 11min, 12min, 13min, 14min, 15min etc.
The preparation method of the Lactobacillus delbrueckii subspecies bulgaricus LB02 bacterial powder provided by the invention is that the bacterial powder is prepared by freeze-drying after high-density fermentation, the method is simple and safe to operate, the bacterial powder yield is high, and the viable count is up to 2 multiplied by 10 11 CFU/g or more.
In a fourth aspect, the present invention provides a conjugated linoleic acid producing strain combination comprising a conjugated linoleic acid producing lactobacillus delbrueckii subsp bulgaricus LB02 and lactobacillus plantarum Lp05 as described in the first aspect;
lactobacillus plantarum (L.) KummerLactobacillus plantarum) Lp05, deposited in China general microbiological culture Collection center (CGMCC) with a deposit number of CGMCC No.23547, a deposit date of 2021, 10 and 09 days, and a deposit address of Beijing Chaoyang area North Chen Xiyu No.1 and 3.
Preferably, the mass ratio of the Lactobacillus delbrueckii subsp bulgaricus LB02 to the Lactobacillus plantarum Lp05 is 1 (0.5-3).
Specific values in the above (0.5-3) are, for example, 0.5, 0.7, 0.9, 1, 1.3, 1.5, 1.7, 2, 2.2, 2.5, 2.8, 3, etc.
In a fifth aspect, the present invention provides the use of a combination of lactobacillus delbrueckii subsp bulgaricus LB02 producing conjugated linoleic acid as described in the first aspect, lactobacillus delbrueckii subsp bulgaricus LB02 powder as described in the second aspect or a strain producing conjugated linoleic acid as described in the fourth aspect for the preparation of a food product comprising conjugated linoleic acid.
Preferably, the food product comprises a liquid beverage or a solid beverage.
In a sixth aspect, the present invention provides a yogurt containing conjugated linoleic acid, wherein the raw materials for preparing the yogurt containing conjugated linoleic acid comprise a fermentation strain, raw milk, linoleic acid, white granulated sugar and a stabilizer;
the fermentation strain comprises Lactobacillus delbrueckii subsp bulgaricus LB02 producing conjugated linoleic acid or the strain combination producing conjugated linoleic acid according to the fourth aspect, and further comprises Streptococcus thermophilus.
Streptococcus thermophilus is an essential strain in yogurt fermentation in the art, and any commercially available or prior art Streptococcus thermophilus strain is available.
Preferably, the preparation raw materials comprise, by mass, 0.001% -0.004% of fermentation strains, 90% -95% of raw milk, 4% -8% of white granulated sugar, 0.1% -1% of stabilizers and 0.05% -1% of linoleic acid.
Specific values among the above 0.001% to 0.004% are, for example, 0.001%, 0.0015%, 0.002%, 0.0025%, 0.003%, 0.0035%, 0.004%, etc.
Specific values in the above 90% -95% are, for example, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95% and the like.
Specific values in the above 4% -8% are, for example, 4%, 5%, 6%, 7%, 8% and the like.
Specific values in the above 0.1% -1% are, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% and the like.
Specific values in the above range from 0.05% to 1% are, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% and the like.
Preferably, the streptococcus thermophilus is 80% -92% by mass, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% etc. in the fermentation strain.
Preferably, the stabilizer comprises any one or a combination of at least two of pectin, agar or soybean polysaccharide, for example, a combination of pectin and agar, a combination of agar and soybean polysaccharide, a combination of pectin and soybean polysaccharide, and the like, and any other combination mode is possible.
In a seventh aspect, the present invention provides a method for preparing a yoghurt rich in conjugated linoleic acid as described in the sixth aspect, the method comprising: mixing raw milk, linoleic acid, white sugar and stabilizer, sterilizing, inoculating fermentation strain, and fermenting.
Preferably, the temperature of the mixing is 30-45deg.C, such as 30deg.C, 32deg.C, 35deg.C, 37deg.C, 40deg.C, 45deg.C, etc., and the time of the mixing is 20-30min, such as 20min, 22min, 24min, 26min, 28min, 30min, etc.
Preferably, the temperature of the fermentation is 40-45 ℃, e.g. 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃ etc., and the time of the fermentation is 6-15 hours, e.g. 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours etc.
In an eighth aspect, the present invention provides a lactic acid bacteria beverage containing conjugated linoleic acid, wherein the preparation raw materials of the lactic acid bacteria beverage containing conjugated linoleic acid comprise skimmed milk powder, white granulated sugar, linoleic acid, citric acid, stabilizer and lactic acid bacteria;
the lactic acid bacteria comprise Lactobacillus delbrueckii subsp bulgaricus LB02 producing conjugated linoleic acid according to the first aspect or a combination of strains producing conjugated linoleic acid according to the fourth aspect, and also comprise Streptococcus thermophilus and Lactobacillus paracasei.
Streptococcus thermophilus is an essential strain in the art for fermentation of lactic acid bacteria beverages, and any commercially available or state of the art Streptococcus thermophilus strain is available. Lactobacillus paracasei is a common strain in the preparation of lactobacillus beverage in the field, plays a role in regulating flavor, and any commercially available or prior art lactobacillus paracasei strain can be used.
Preferably, in the lactic acid bacteria beverage, the inoculation amount of the lactic acid bacteria is 20-40g/T, such as 20g/T, 22g/T, 25g/T, 27g/T, 30g/T, 32g/T, 35g/T, 37g/T, 40g/T, etc.
In a ninth aspect, the present invention provides a method for preparing a conjugated linoleic acid-containing lactic acid bacteria beverage according to the eighth aspect, comprising the steps of:
(1) Mixing skimmed milk powder, part of white sugar, and linoleic acid with water, sterilizing, inoculating lactobacillus, and fermenting to obtain fermented milk;
(2) Mixing fermented milk, residual white sugar and stabilizer to obtain mixed solution, and adding citric acid to regulate acidity.
Preferably, the portion of white granulated sugar comprises 10% -50% of the total mass of white granulated sugar, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% and the like.
Preferably, the temperature of the mixing in step (1) is 40-50 ℃, e.g. 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, etc., and the time of the mixing is 20-30min, e.g. 20min, 22min, 24min, 26min, 28min, 30min, etc.
Preferably, the fermentation temperature is 37-40 ℃, such as 37 ℃, 37.5 ℃, 38 ℃, 38.5 ℃, 39 ℃, 39.5 ℃,40 ℃, etc., and the fermentation time is 15-48 hours, such as 15 hours, 18 hours, 20 hours, 24 hours, 28 hours, 30 hours, 34 hours, 38 hours, 40 hours, 44 hours, 48 hours, etc.
Preferably, the fermented milk in the step (1) comprises 10-20% of skimmed milk powder, 4-8% of white granulated sugar, 0.05-1% of linoleic acid and the balance of water in percentage by mass.
Specific values in the above 10% -20% are, for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.
Specific values in the above 4% -8% are, for example, 4%, 5%, 6%, 7%, 8% and the like.
Specific values in the above range from 0.05% to 1% are, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% and the like.
Preferably, the mixed solution in the step (2) comprises 10-25% of fermented milk, 2-8% of white granulated sugar, 0.2-0.8% of stabilizer and the balance of water in percentage by mass.
Specific values in the above 10% -25% are, for example, 10%, 12%, 15%, 17%, 20%, 22%, 25% and the like.
Specific values in the above 2% -8% are, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8% and the like.
Specific values in the above 0.2% to 0.8% are, for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% and the like.
Preferably, the stabilizer comprises any one or a combination of at least two of pectin, agar or soybean polysaccharide, for example, a combination of pectin and agar, a combination of agar and soybean polysaccharide, a combination of pectin and soybean polysaccharide, and the like, and any other combination mode is possible.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a novel strain for producing conjugated linoleic acid: the lactobacillus delbrueckii subspecies bulgaricus LB02 can convert linoleic acid into conjugated linoleic acid, the conversion rate is high, the method is important for industrial production of biological conversion of conjugated linoleic acid, and the preparation method of the lactobacillus delbrueckii subspecies bulgaricus LB02 bacterial powder is formed by adopting high-density fermentation and freeze-drying, and the method is simple and safe to operate, and the bacterial powder yield and the viable count are high.
(2) The yoghourt and the lactobacillus beverage prepared by the LB02 strain provided by the invention have high conjugated linoleic acid content, have important health care functions, such as immunity improvement, antioxidation, anti-tumor, atherosclerosis resistance, weight reduction and the like, are beneficial to human health, and meet the demands of most modern people on the functionality of the yoghourt and the lactobacillus beverage.
(3) The invention creatively discovers that lactobacillus plantarum Lp05 and lactobacillus delbrueckii subspecies bulgaricus LB02 mutually promote and have a synergistic effect in the aspect of producing conjugated linoleic acid, and the conjugated linoleic acid content in a fermentation product is obviously improved by using the lactobacillus plantarum Lp05 and the lactobacillus delbrueckii subspecies bulgaricus LB02 after being compounded for fermentation.
Detailed Description
In order to further describe the technical means adopted by the present invention and the effects thereof, the following describes the technical scheme of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
In the following examples, reagents and consumables were purchased from the manufacturers of reagents as conventional in the art unless otherwise specified; unless otherwise indicated, all methods and techniques used are those conventional in the art.
The following examples relate to the following raw material information:
raw cow milk was purchased from the company gmbh, inc;
skim milk powder was purchased from New Zealand constant Natural group Co., ltd;
linoleic acid was purchased from the wuhan bailing organism;
gram staining reagents were purchased from beijing solebao biotechnology limited;
the soybean polysaccharide and pectin were purchased from Jinan quankang biotechnology limited.
Example 1
Screening and identification of Lactobacillus delbrueckii subspecies bulgaricus LB02
(1) Isolation and purification of strains
Taking 2g of traditional fermented yogurt sample (from Sichuan Cuminum cyminum), adding into 18mL of sterile water, mixing, homogenizing, sequentially diluting 1mL of bacterial liquid into 9mL of sterile water to make the sample concentration be diluted to 10 in gradient 5 And (3) respectively coating 100 mu L of each diluted bacterial suspension on an MRS solid culture medium, culturing at 37 ℃ for 48 hours, selecting a proper single colony, inoculating the single colony into the MRS liquid culture medium after 3 generations of purification, culturing at 37 ℃ for 18 hours to obtain bacterial liquid, mixing with 40% of glycerol 1:1, and preserving at a low temperature of-80 ℃.
(2) Screening of conjugated linoleic acid-producing Strain
Inoculating the strain glycerol tube preserved at low temperature into MRS liquid culture medium, activating for 2 times at 37 ℃, culturing for 15 hours each time, inoculating into MRS liquid culture medium containing 0.5% linoleic acid according to 2% inoculum size, standing and culturing for 48h at 37 ℃ and centrifuging for 5min at 6000rpm to obtain culture solution. Extracting conjugated linoleic acid from the culture solution, and detecting the content by ultraviolet spectrophotometry, wherein the strain with the number LB02 has the highest conjugated linoleic acid content of about 1.67mg/mL.
(3) Morphological observation and physicochemical experiments of strains
The bacterial colony morphology of the strain LB02 plate and the morphological characteristics of gram-stained cells are carefully observed and recorded, and a catalase test, a contact enzyme test and a carbon source acidogenesis (API 50 CH) test are simultaneously carried out, so that the bacterial colony morphology of the strain on an MRS culture medium is irregular snowy, pale white and has a matte surface, the bacterial cells under a microscope are in a short rod shape, the bacterial cell rough glaze is uneven, and the bacterial colony is gram-positive, catalase test negative and contact enzyme test negative.
(4) Identification of species
The strain LB02 is Lactobacillus delbrueckii subspecies bulgaricus, and the 16S rDNA gene sequence is determined as follows (SEQ ID NO. 1):
ATCATGCTTATCGTAACTGTCTGCTTAGGCGGCTGACTCCTATAAAGGTTATCCCACCGACTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAG
AACAGCTTTAAGAGATCCGCTTACCCTCGCGGGTTCGCTTCTCGTTGTACT
GCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGA
CGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCTTTAGAGTGCC
CAACTTAATGATGGCAACTAAAGACAAGGGTTGCGCTCGTTGCGGGACTT
AACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCT
CTGCGTCCCCGAAGGGAACCACCTATCTCTAGGTGTAGCACAGGATGTCA
AGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACC
GCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTAC
TCCCCAGGCGGAGCGCTTAATGCGTTTGCTGCGGCACTGAGAACCGGAAA
GTCCCCAACACCTAGCGCTCATCGTTTACGGCATGGACTACCAGGGTATCT
AATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAG
AGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCT
ACACATGGAATTCCACTCTCCTCTTCTGCACTCAAGAATGACAGTTTCCGA
TGCAGTTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTATCATTCCGC
CTGCGCTCGCTTTACGCCCAATAAATCCCGGACAACGCTTGCCACCTACGT
ATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTCTGGTTGATTACCG
TCAAATAAAGACCAGTTACTGCCTCTATCCTTCTTCACCAACAACAGAGCT
TTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTG
CGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCG
TGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTACGCATCAT
TGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATGCGCCGCGGGCTCAT
CCTAAAGTGACAGCTTACGCCGCCTTTCAAACTTGAATCATGCGATTCATG
TTGTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGTCTTTAGGGC
AGATTGCCCACGTGTTACTCACCCATCCGCCGCTAGCGTCCAACAAATCAC
CCCGAAGGGATCTTTGAATTCAGCTCGCTCGACTTGCATGTATAGCACGCCGCCCAGTGCCAA。
example 2
(1) Preparation of Lactobacillus delbrueckii subspecies bulgaricus LB02 freeze-dried bacterial powder
The lactobacillus delbrueckii subspecies bulgaricus LB02 is subjected to high-density fermentation culture to obtain bacterial powder with higher viable count, and the specific operation steps are as follows:
inoculating LB02 strain preserved by glycerol pipe into seed culture medium according to 2% ratio, standing at 37deg.C for 16h, and activating for 2 generations. Inoculating the activated seed culture solution into a fermentation culture medium, and culturing for 15h at 37 ℃ with the inoculum size of 3%, wherein the pH is controlled to be 6.2, thus obtaining LB02 bacterial solution.
Centrifuging the bacterial liquid at 6000r/min for 10min, removing supernatant, collecting bacterial mud, and adding a mixed protective agent comprising trehalose, maltodextrin, sucrose and tween-80, wherein the protective agent comprises the following components in percentage by mass: 25% trehalose, 5% maltodextrin, 3% sucrose and 0.1% Tween-80, the balance being water. The mass ratio of the protective agent to the bacterial mud is 1:1, and then the LB02 freeze-dried bacterial powder is prepared after vacuum freeze-drying, wherein the viable count in the bacterial powder is 2 multiplied by 10 11 CFU/g。
The formula of the seed culture medium comprises: glucose 20g/L, lactose 10g/L, peptone 10g/L, beef extract 5g/L, yeast extract 4g/L, K 2 HPO 4 2g/L, diammonium hydrogen citrate 2g/L, tween-80.0 g/L, mgSO 4 0.5g/L、MnSO 4 0.2g/L。
The fermentation medium comprises the following components in percentage by weight: glucose 30g/L, lactose 10g/L, peptone 15g/L, beef extract 8g/L, yeast extract 7g/L, K 2 HPO 4 2g/L, 2g/L diammonium hydrogen citrate, 4g/L sodium acetate, and Tween-80.0 g/L, mgSO 4 0.5g/L、CaCl 2 0.3g/L、MnSO 4 0.1g/L。
(2) Preparation of lactobacillus plantarum Lp05 freeze-dried bacterial powder
Lactobacillus plantarum (Lactobacillus plantarum) Lp05 strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.23547, a preservation date of 2021 and 10 month 09 days, and a preservation address of North Chen Xiyu No.1 and 3 in the Chaoyang area of Beijing city.
After a lactobacillus plantarum Lp05 freezing tube is dissolved, inoculating the lactobacillus plantarum Lp05 freezing tube into an MRS liquid culture medium according to 2%, and culturing the lactobacillus plantarum Lp05 freezing tube at 37 ℃ for 16 hours to obtain Lp05 bacterial liquid, collecting bacterial mud and preparing freeze-dried bacterial powder, wherein the preparation of the freeze-dried bacterial powder refers to (1) lactobacillus delbrueckii subspecies bulgaricus LB02 freeze-dried bacterial powder.
(3) Preparation of lyophilized powder of Lactobacillus delbrueckii subspecies bulgaricus ATCC 11842 reference (1) preparation of lyophilized powder of Lactobacillus delbrueckii subspecies bulgaricus LB 02.
(4) Preparation of Lactobacillus plantarum ATCC 8014 lyophilized powder reference (2) preparation of Lactobacillus plantarum Lp05 lyophilized powder.
(5) Grouping freeze-dried bacterial powder:
group 1: lactobacillus delbrueckii subspecies bulgaricus LB02 lyophilized powder;
group 2: lactobacillus plantarum Lp05 freeze-dried powder;
group 3: mixing lactobacillus delbrueckii subsp bulgaricus LB02 freeze-dried bacterial powder and lactobacillus plantarum Lp05 freeze-dried bacterial powder in a mass ratio of 2:1;
group 4: mixing lactobacillus delbrueckii subsp bulgaricus LB02 freeze-dried bacterial powder and lactobacillus plantarum Lp05 freeze-dried bacterial powder in a mass ratio of 3:1;
group 5: mixing lactobacillus delbrueckii subsp bulgaricus LB02 freeze-dried bacterial powder and lactobacillus plantarum Lp05 freeze-dried bacterial powder in a mass ratio of 1:1;
group 6: mixing lactobacillus delbrueckii subsp bulgaricus LB02 freeze-dried bacterial powder and lactobacillus plantarum ATCC 8014 freeze-dried bacterial powder in a mass ratio of 2:1;
group 7: the lactobacillus delbrueckii subspecies bulgaricus ATCC 11842 freeze-dried bacterial powder and the lactobacillus plantarum Lp05 freeze-dried bacterial powder are mixed in a mass ratio of 2:1.
Example 3
Preparation of functional yoghurt rich in conjugated linoleic acid
After mixing the same mass of each group of the bacterial powders provided in example 2 with the streptococcus thermophilus ST81 bacterial powder (commercial products, from micro Kang Yisheng bacteria (su state) stock, purchase website: https:// www.ingredientsonline.com/cataogsearch/result/:
(1) Adding white granulated sugar and stabilizer into raw milk, dissolving thoroughly, adding sterile linoleic acid filtered by 0.22 μm filter membrane, and hydrating at 40deg.C for 25min to obtain fermentation base material. Wherein the weight percentage is as follows: white granulated sugar accounting for 6 percent, stabilizer (pectin and soybean polysaccharide accounting for 1:1) accounting for 0.5 percent, linoleic acid accounting for 0.5 percent, and the balance being cow milk;
(2) Sterilizing the hydrated fermentation base material at 95 ℃ for 10min, and then rapidly cooling to 40 ℃ in cold water;
(3) And (3) respectively mixing the bacterial powders of each group provided in the example 2 with the streptococcus thermophilus bacterial powder according to the mass ratio of 1:5, inoculating the mixed bacterial powder into the sterilized fermentation base material according to the total inoculation amount of 30g/T, fermenting at the temperature of 43 ℃ for 12 hours, stopping fermenting when the acidity reaches 80 DEG T, pH 4.48.48, and refrigerating at the temperature of 4 ℃ for overnight to obtain the finished product.
Example 4
Preparation of lactobacillus beverage rich in conjugated linoleic acid
Lactobacillus delbrueckii subsp bulgaricus LB02 freeze-dried bacterial powder, streptococcus thermophilus ST81 bacterial powder and lactobacillus paracasei bacterial powder (ATCC 25302, lactobacillus paracasei is a common bacterial strain in the preparation of beverages in the field, and the lactobacillus paracasei related to the embodiment does not have the function of producing conjugated linoleic acid) are compounded and fermented, and then the beverage is prepared by the specific steps of:
(1) Hydrating skimmed milk powder, white sugar and sterile linoleic acid filtered by 0.22 μm filter membrane at 45deg.C for 30min, wherein the concentration of skimmed reconstituted milk is 20%, the content of skimmed reconstituted milk is 93.5%, the content of white sugar is 6%, and the content of linoleic acid is 0.5%;
(2) Sterilizing the hydrated fermentation base material at 95 ℃ for 10min, and then rapidly cooling to 40 ℃ in cold water;
(3) Inoculating lactobacillus delbrueckii subsp bulgaricus LB02 freeze-dried bacterial powder, streptococcus thermophilus bacterial powder and lactobacillus paracasei bacterial powder into the sterilized fermentation base material according to the mass ratio of 1:5:1, wherein the total inoculation amount is 30g/T, the fermentation temperature is 37 ℃, the time is 48 hours, and the acidity reaches 200 ℃ and then the cold storage is carried out at 4 ℃ for later use;
(4) Dissolving pectin and soybean polysaccharide with 70 ℃ pure water, stirring uniformly, preparing a stabilizer aqueous solution with the concentration of 0.5% (the ratio of pectin to soybean polysaccharide is 1:2), adding 5% of white granulated sugar, adding 20% of fermented milk obtained in the step (3), finally adding citric acid to adjust the pH to 70 ℃, homogenizing under 20MPa, and cooling to 25 ℃ after pasteurization to obtain the lactobacillus beverage rich in conjugated linoleic acid.
Test case
Determination of conjugated linoleic acid content
The conjugated linoleic acid content in each group of yoghurt of example 3 and the lactic acid bacteria beverage of example 4 was determined as follows:
(1) Drawing a standard curve:
conjugated linoleic acid is prepared into gradient solutions with the concentration of 0mg/mL, 2mg/mL, 4mg/mL, 6mg/mL, 8mg/mL, 10mg/mL, 12mg/mL and 14mg/mL by taking normal hexane as a solvent. N-hexane is used as a reference solution, colorimetric is carried out at 234nm wavelength, the absorbance is recorded and a standard curve is drawn.
(2) Determination of conjugated linoleic acid content in the sample:
taking 0.5mL of a sample to be detected in a centrifuge tube, adding 1.5mL of isopropanol, and shaking uniformly. Adding 1.5mL of n-hexane, and shaking uniformly; then, 1.5mL of distilled water was added, the mixture was shaken well, and the mixture was centrifuged at 4000r/min for 10min. The supernatant was taken and the volume was set to 10mL. N-hexane is used as a reference solution, colorimetric is carried out at 234nm wavelength, the absorbance is recorded, and the corresponding conjugated linoleic acid content is calculated according to a standard curve.
The results are shown in Table 1.
TABLE 1
The results show that: the dairy products such as yoghourt and the like prepared by fermenting lactobacillus delbrueckii subspecies bulgaricus LB02 for producing conjugated linoleic acid provided by the invention are rich in conjugated linoleic acid.
In particular, as is clear from the comparison results among groups 1, 2, 3, 6 and 7, although the capacity of the lactobacillus plantarum Lp05 for producing conjugated linoleic acid is not as good as that of lactobacillus delbrueckii bulgaricus strain LB02, under the condition that the total amount is unchanged, the content of conjugated linoleic acid in the product obtained by fermenting the lactobacillus plantarum Lp05 and the lactobacillus delbrueckii bulgaricus strain LB02 after the lactobacillus delbrueckii bulgaricus strain is obviously higher than that of the product obtained by fermenting the lactobacillus delbrueckii bulgaricus strain, the result shows that the lactobacillus plantarum Lp05 and the lactobacillus delbrueckii bulgaricus strain mutually promote, and the lactobacillus delbrueckii bulgaricus strain has a synergistic effect in terms of producing the conjugated linoleic acid, but the effect is not seen, and the yield of the conjugated linoleic acid is not ideal.
The applicant states that the present invention is illustrated by the above examples as a conjugated linoleic acid producing lactobacillus delbrueckii subsp bulgaricus LB02, bacterial powder, bacterial strain combination and applications thereof, but the present invention is not limited to the above examples, i.e. it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
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 scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. Lactobacillus delbrueckii subsp bulgaricus producing conjugated linoleic acidLactobacillus delbrueckii subsp bulgaricus) The method is characterized in that the Lactobacillus delbrueckii subspecies bulgaricus LB02 is preserved in China general microbiological culture Collection center (CGMCC) No.24664, the preservation date is 2022, 4 months and 11 days, and the preservation address is North Chen West Lu No.1, 3 of the Korean region of Beijing city.
2. A lactobacillus delbrueckii subspecies bulgaricus LB02 powder, wherein the lactobacillus delbrueckii subspecies bulgaricus LB02 powder comprises the lactobacillus delbrueckii subspecies bulgaricus LB02 producing conjugated linoleic acid according to claim 1 and a protective agent.
3. A process for preparing lactobacillus delbrueckii subsp bulgaricus LB02 powder as claimed in claim 2, comprising: culturing Lactobacillus delbrueckii subspecies bulgaricus LB02 to obtain culture solution, centrifuging, collecting bacterial mud, mixing with protective agent, and lyophilizing.
4. A conjugated linoleic acid-producing strain combination comprising the conjugated linoleic acid-producing Lactobacillus delbrueckii subsp. Bulgaricus LB02 and Lactobacillus plantarum strain of claim 1Lactobacillus plantarum) Lp05, wherein the preservation number of the lactobacillus plantarum Lp05 is CGMCC No.23547.
5. The combination of conjugated linoleic acid producing strains according to claim 4, wherein the mass ratio of lactobacillus delbrueckii subsp. Bulgaricus LB02 to lactobacillus plantarum Lp05 is 1 (0.5-3).
6. Use of a combination of lactobacillus delbrueckii subsp bulgaricus LB02 producing conjugated linoleic acid as defined in claim 1, lactobacillus delbrueckii subsp bulgaricus LB02 powder as defined in claim 2 or a strain producing conjugated linoleic acid as defined in any of claims 4 to 5 for the preparation of a food product comprising conjugated linoleic acid.
7. The yoghourt containing the conjugated linoleic acid is characterized in that the preparation raw materials of the yoghourt containing the conjugated linoleic acid comprise a fermentation strain, raw milk, linoleic acid, white granulated sugar and a stabilizer;
the fermentation strain comprises the Lactobacillus delbrueckii subsp bulgaricus LB02 producing conjugated linoleic acid or the strain combination producing conjugated linoleic acid as set forth in any one of claims 4 to 5, and the fermentation strain further comprises streptococcus thermophilus @Streptococcus thermophilus）。
8. A process for preparing a yoghurt containing conjugated linoleic acid as claimed in claim 7, which comprises: mixing raw milk, linoleic acid, white sugar and stabilizer, sterilizing, inoculating fermentation strain, and fermenting.
9. The lactic acid bacteria beverage containing the conjugated linoleic acid is characterized in that the preparation raw materials of the lactic acid bacteria beverage containing the conjugated linoleic acid comprise skimmed milk powder, white granulated sugar, linoleic acid, citric acid, a stabilizer and lactic acid bacteria;
the lactic acid bacteria comprising the combination of Lactobacillus delbrueckii subsp bulgaricus LB02 producing conjugated linoleic acid as defined in claim 1 or the strain producing conjugated linoleic acid as defined in any one of claims 4 to 5, and further comprising Streptococcus thermophilus and Lactobacillus paracasei @Lactobacillus paracasei）。
10. A method for preparing a conjugated linoleic acid containing lactic acid bacteria beverage according to claim 9, wherein the method comprises the steps of:
(1) Mixing skimmed milk powder, part of white sugar, and linoleic acid with water, sterilizing, inoculating lactobacillus, and fermenting to obtain fermented milk;
(2) Mixing fermented milk, residual white sugar and stabilizer to obtain mixed solution, and adding citric acid to regulate acidity.
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