CN112625971A

CN112625971A - Lactobacillus delbrueckii capable of consuming a large amount of lactose and resisting acid, bile salt and oxidation and application thereof

Info

Publication number: CN112625971A
Application number: CN202011643471.6A
Authority: CN
Inventors: 李理; 周晴晴; 李言郡; 陈苏; 余腾斐; 李宝磊; 朱珺
Original assignee: HANGZHOU WAHAHA TECHNOLOGY CO LTD
Current assignee: HANGZHOU WAHAHA TECHNOLOGY CO LTD
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-04-09
Anticipated expiration: 2040-12-30
Also published as: CN112625971B

Abstract

The invention relates to the field of microbial fermentation and discloses a strain which can consume a large amount of lactoseLactobacillus delbrueckii WHH3887 has the preservation number of CGMCC No. 20090; the microorganism classification is named as Lactobacillus delbrueckii subspecies bulgaricusLactobacillus delbrueckii subsp.bulgaricus. The strain can obviously reduce the lactose content in raw milk after fermentation, and has good fermentation performance. The strain is applied or compounded with other starter strains to produce fermented milk, and the requirement of zero lactose (less than or equal to 0.5g/100g) can be achieved. In addition, compared with the similar strains, the strain has more excellent acid resistance, cholate resistance and oxidation resistance, and has stronger applicability.

Description

Lactobacillus delbrueckii capable of consuming a large amount of lactose and resisting acid, bile salt and oxidation and application thereof

Technical Field

The invention relates to the field of microbial fermentation, in particular to lactobacillus delbrueckii capable of consuming a large amount of lactose and having acid resistance, cholate resistance and oxidation resistance and application thereof.

Background

Lactose is a specific carbohydrate in mammalian milk and is closely related to human health. The lactose content in cow milk is 4.7%, and the lactose content in human milk is about 7%. Lactose is completely dissolved in milk and is the most stable component in cow milk. Lactase activity in humans decreases with age and, after adults, is only 5-10% of normal infant levels. After lactose in food enters the small intestine, lactose is not decomposed into monosaccharides (glucose and galactose) and is absorbed due to lactase deficiency, and lactose indigestion (lactose indigestion) and lactose malabsorption (lactose malabsorption) are formed. When lactose enters colon, it is fermented by bacteria to produce short chain organic acids such as acetic acid and propionic acidButyric acid, etc. and gases, e.g. methane, H₂、CO₂Etc., causing borborygmus, abdominal pain, rectal gas and osmotic diarrhea, which are known as lactose intolerance (LD). According to the investigation, the incidence rate of LD of Chinese Han adults is 75-95%, and the incidence rate of LD of minority nationalities is 76-95.5%. Lactose malabsorption can produce a range of chronic effects, the most important of which is the effect on nutritional status. Lactose intolerant people often drink no or little milk to avoid intolerance symptoms, so that the absorption of high-quality calcium is greatly reduced, and osteoporosis (old people) and slow growth (children and teenagers) are commonly shown.

In order to eliminate the adverse effect of lactose on intolerants and improve the intake of dairy products, researchers at home and abroad do a lot of work, and oral administration of lactic acid bacteria and drinking of low-lactose dairy products are proposed to effectively relieve the symptoms of lactose intolerance. Many methods for preparing low lactose dairy products include ultrafiltration concentration, enzymatic decomposition, lactic acid bacteria fermentation, and the like. The technology for preparing low lactose milk by enzymatic decomposition is applied to products, generally, lactase is subjected to solid phase solidification and then is filled into a reaction column, raw milk is injected from one section of the column and flows out from the other end after contacting with the enzyme. The dairy products produced by applying the technology in China at present are small in scale and relatively dispersed, and in addition, the investment cost in the early stage is high, the technical difficulty is high, and the popularization is difficult.

Compared with the enzymolysis method, the method for preparing the low-lactose fermentation by fermenting the raw material milk with the lactobacillus has the advantages of easy operation, easy popularization, no additional increase of large cost and the like. One of the main mechanisms of the lactic acid bacteria for relieving lactose intolerance is that the thalli generate beta-galactosidase (lactase) in the fermentation process, and the beta-galactosidase hydrolyzes lactose into glucose and galactose which can be utilized by human bodies, so that the lactose content in raw milk is reduced, and the lactose malabsorption and the lactose intolerance are reduced. After the cow milk is fermented by common starter strains, the lactose content is reduced to a certain degree, but a considerable part of the lactose is still remained in the cow milk, and the claimed requirement (less than or equal to 0.5g/100g) of zero lactose in GB28050-2011 national food safety Standard-general rules for prepackaged food Nutrition labels is difficult to meet under the condition of not adding lactase. Thus, people with low lactose tolerance still develop intolerance symptoms after drinking. The method has the advantages that the starter strains capable of consuming a large amount of lactose are obtained through screening, the method for preparing the low-lactose or zero-lactose fermented milk has wide market prospect, and the method has important practical significance for improving the milk product drinking amount of lactose intolerant people and solving the health problems of Chinese people.

Disclosure of Invention

In order to solve the technical problems, the invention provides the lactobacillus delbrueckii strain which can consume a large amount of lactose and has acid resistance, cholate resistance and oxidation resistance and the application thereof. The strain is applied or compounded with other starter strains to produce fermented milk, and the lactose residual quantity can reach the zero lactose claim requirement (less than or equal to 0.5g/100g) in GB28050-2011 national standard for food safety-general rules of prepackaged food nutrition labels). The strain can overcome the problem that some consumers are intolerant to lactose due to the fact that fermented milk drinks in the prior art contain high lactose, and the problem of dairy consumption of lactose intolerant consumers is solved to a certain extent. In addition, compared with the similar strains, the strain has more excellent acid resistance, cholate resistance and oxidation resistance, and has stronger applicability.

The specific technical scheme of the invention is as follows:

the invention provides a Lactobacillus delbrueckii strain which can consume a large amount of lactose and has acid resistance, cholate resistance and oxidation resistance, is named as WHH3887, is obtained by separating the Lactobacillus delbrueckii strain from traditional yoghourt in Tibet areas of China in earlier work, is preserved in China general microbiological culture Collection center (CGMCC) at 6-15 days in 2020, and has the preservation number of CGMCC No. 20090; the microorganism is classified and named Lactobacillus delbrueckii subsp.

The lactobacillus delbrueckii subsp bulgaricus 3887 strain provided by the invention has the capacity of consuming a large amount of lactose, can obviously reduce the lactose content in raw milk in a shorter time compared with other strains, and can further reduce the lactose content after a prolonged time, but the production cost can be obviously reduced due to the shorter fermentation time, and meanwhile, the problems of poor quality and flavor of finished yoghurt and the like caused by unstable acid after the strain are reduced, so that the capacity of reducing lactose in a short time of the strain is emphasized.

The single strain fermentation can form fermented milk with good sensory quality, the surface is flat, a part of yogurt is scooped by a spoon, the cut surface is neat and smooth, and the fermented milk has typical and pleasant fermentation taste and smell. After the direct vat set starter of the strain is fermented for 24 hours at 40 ℃ in a skim milk base, the residual amount of lactose in the fermented milk is lower than 0.5g/100g, and the obtained fermented milk has good texture, flavor and taste.

On the other hand, the Lactobacillus delbrueckii subspecies bulgaricus WHH3887 strain provided by the invention has better acid resistance and cholate resistance. The acid resistance is shown in that after the strain WHH3887 is cultured in a culture medium with the pH value of 4.0 for 12 hours, the survival rate is 81.4 percent, and after the strain is cultured in the culture medium with the pH value of 3.0 for 12 hours, the survival rate is obviously higher than that of a control strain. The characteristic of the bile salt resistance is shown in that after the strain WHH3887 is cultured in a culture medium containing 0.5 percent of the bovine bile salt for 12 hours, the survival rate is 59.30 percent and is obviously higher than that of a control strain.

In addition, the Lactobacillus delbrueckii subspecies bulgaricus WHH3887 strain provided by the invention has antioxidant property, and is mainly characterized by excellent H₂O₂Tolerance, hydroxyl radical scavenging, and DPPH radical scavenging.

In a second aspect, the present invention provides a mutant of lactobacillus delbrueckii, which can consume a large amount of lactose and has acid resistance, bile salt resistance and oxidation resistance, wherein the mutant is obtained by subjecting the lactobacillus delbrueckii to mutagenesis, domestication, genetic recombination or natural mutation.

Preferably, the mutant has a nucleotide sequence at least 90% homologous to the lactobacillus delbrueckii strain, and the mutant has at least 90% or more of lactose decomposition ability, acid resistance, bile salt resistance and oxidation resistance, respectively, to the lactobacillus delbrueckii strain.

Further, the mutant has a nucleotide sequence at least 95% homologous with the lactobacillus delbrueckii, and the mutant has at least 95% or more of lactose decomposition capability, acid resistance, bile salt resistance and oxidation resistance with the lactobacillus delbrueckii respectively.

Still further, the mutant has a nucleotide sequence having at least 99% homology with the lactobacillus delbrueckii, and the mutant has at least 99% or more of lactose decomposition ability, acid resistance, bile salt resistance and oxidation resistance with the lactobacillus delbrueckii, respectively.

In a third aspect, the present invention provides a bacterial culture comprising said Lactobacillus delbrueckii or comprising said mutant.

Preferably, the bacterial culture is a bacterial solution or a bacterial agent.

In a fourth aspect, the invention provides an application of the lactobacillus delbrueckii or the mutant or the thallus culture in preparing a lactobacillus direct vat set starter.

In a fifth aspect, the invention provides an application of the lactobacillus delbrueckii, the streptococcus thermophilus 445 and the lactococcus lactis 2353 in preparation of a lactobacillus direct vat set starter after compounding.

Preferably, the compounding ratio of the lactobacillus delbrueckii to the streptococcus thermophilus 445 and the lactococcus lactis 2353 is (0.8-1.2) to (0.8-1.2).

In a sixth aspect, the invention provides an application of the lactobacillus direct vat set starter in preparation of low-lactose fermented milk, zero-lactose fermented milk, normal fermented milk, low-lactose lactobacillus beverage, zero-lactose lactobacillus beverage and normal lactobacillus beverage.

In a seventh aspect, the invention provides an application of the lactobacillus direct vat set starter in preparing foods, beverages, health foods and medicines for improving lactose malabsorption, lactose dyspepsia and lactose intolerance.

Compared with the prior art, the invention has the following technical effects:

(1) the Lactobacillus delbrueckii subspecies bulgaricus WHH3887 strain provided by the invention can form fermented milk with good texture and flavor after fermentation, and the fermented milk is particularly characterized in that the surface is flat, a part of yogurt is scooped by a spoon, the cut surface is neat and smooth, and the fermented milk has typical and pleasant fermentation taste and smell.

(2) The Lactobacillus delbrueckii subspecies Bulgaria WHH3887 strain provided by the invention can obviously reduce lactose in a short time by using a large amount of lactose in raw milk of intracellular lactase in the fermentation process. After fermentation at 40 ℃ for 24h, the lactose content in the fermented milk reaches the zero lactose claim requirement (less than or equal to 0.5g/100g) in GB28050-2011 national standard for food safety-general rules of prepackaged food nutrition labels. Meanwhile, the formed fermented milk has typical and pleasant fermentation aroma, and is fine and smooth in taste.

(3) The Lactobacillus delbrueckii subspecies bulgaricus WHH3887 strain provided by the invention has good acid resistance, cholate resistance and oxidation resistance.

(4) The Lactobacillus delbrueckii subspecies Bulgaria WHH3887 strain provided by the invention has good fermentation adaptability, can be fermented cooperatively with other starter strains to prepare low-lactose fermented milk products with different textures and flavors, and can also be matched with probiotic strains to prepare low-lactose fermented milk products with probiotic functions.

(5) Compared with a lactase method, the method for preparing the low-lactose or zero-lactose fermented dairy product by using the Lactobacillus delbrueckii subspecies Bulgaria WHH3887 strain has the advantages of low cost, small technical difficulty, easy operation and the like.

Drawings

FIG. 1 is a liquid chromatogram peak diagram of a mixed sugar standard, wherein the standard is a mixed solution of a fructose standard, a glucose standard, a sucrose standard, a maltose standard and a lactose standard, and the retention time of the lactose standard is 13.480min, so that the lactose standard can be used for detecting the qualitative index of lactose in a sample.

Fig. 2 is a standard curve with lactose standard substance concentration as abscissa and peak area values obtained by liquid chromatography detection of standard substance solutions with different concentrations as ordinate, which can be used for quantitative index of lactose in detection samples.

FIG. 3 is a microbiological morphology observation chart of the strain of the present invention, in which panel A is a cell morphology observed under a 100-fold optical microscope after gram staining; and the graph B is the colony morphology after the MRS solid culture medium grows for 48-72 h.

FIG. 4 is a graph showing fermentation acid production curves of the bacterial strain and the compound formula thereof applied to reconstituted skim milk.

Detailed Description

The present invention will be further described with reference to the following examples.

General examples

The lactobacillus delbrueckii strain which can consume a large amount of lactose and has acid resistance, cholate resistance and oxidation resistance is named as WHH3887 and is preserved in China general microbiological culture Collection center (CGMCC) 6-15 days in 2020 with the preservation number of CGMCC No. 20090; the microorganism is classified and named Lactobacillus delbrueckii subsp.

A mutant of lactobacillus delbrueckii which can consume a large amount of lactose and has acid resistance, cholate resistance and oxidation resistance, wherein the mutant is obtained by performing mutagenesis, domestication, gene recombination or natural mutation on the lactobacillus delbrueckii.

Preferably, the mutant has a nucleotide sequence at least 90% homologous to the lactobacillus delbrueckii strain, and the mutant has at least 90% or more of lactose decomposition ability, acid resistance, bile salt resistance and oxidation resistance, respectively, to the lactobacillus delbrueckii strain. Further, the mutant has a nucleotide sequence at least 95% homologous with the lactobacillus delbrueckii, and the mutant has at least 95% or more of lactose decomposition capability, acid resistance, bile salt resistance and oxidation resistance with the lactobacillus delbrueckii respectively. Still further, the mutant has a nucleotide sequence having at least 99% homology with the lactobacillus delbrueckii, and the mutant has at least 99% or more of lactose decomposition ability, acid resistance, bile salt resistance and oxidation resistance with the lactobacillus delbrueckii, respectively.

A bacterial cell culture containing the Lactobacillus delbrueckii or the mutant. Preferably, the bacterial culture is a bacterial solution or a bacterial agent.

The Lactobacillus delbrueckii or the mutant or the thallus culture is applied to the preparation of a lactobacillus direct vat set starter.

The lactobacillus delbrueckii is compounded with streptococcus thermophilus 445 and lactococcus lactis 2353 and then applied to preparation of the lactobacillus direct vat set starter. The compounding ratio of the lactobacillus delbrueckii, the streptococcus thermophilus 445 and the lactococcus lactis 2353 is (0.8-1.2) to (0.8-1.2).

The lactobacillus direct vat set starter is applied to preparing low-lactose fermented milk, zero-lactose fermented milk, common fermented milk, low-lactose lactobacillus beverage, zero-lactose lactobacillus beverage and common lactobacillus beverage.

The lactobacillus direct vat set starter is applied to the preparation of foods, beverages, health-care foods and medicines aiming at improving lactose malabsorption, lactose dyspepsia and lactose intolerance.

Example 1

The strain capable of consuming a large amount of lactose is screened primarily by activating the strain preserved in glycerol with MRS + 2% lactose liquid medium, and is cultured overnight at 37 ℃ in a static way. And (4) continuously passaging twice, centrifuging the bacterial suspension to remove supernatant, and resuspending bacterial sludge by 5mL of skim milk. Inoculating into 200mL sterilized skim milk according to the proportion of 2% (volume ratio), standing and fermenting at 40 ℃ for 24h, demulsifying to terminate fermentation, measuring acidity and recording the sensory quality of the fermented milk. Sensory evaluation of fermented milk was divided into 4 grades, a etc.: good curd can be formed, milk white is obtained, whey is less, and pleasant aroma is generated; b and the like: good curd can be formed, milk white is milk white, whey is more or less layered, and pleasant fragrance is generated but the smell is weaker; c and the like: half curd or curd is not firm enough, has more whey or is obvious in layering, and has no fragrance or peculiar smell; d, and the like: no curd can be formed or off-flavors can be generated.

78 food-grade lactic acid bacteria strains included in a bacterial list available for food, issued by the ministry of health in 2010, were preliminarily screened for acid production and sensory quality, and 26 strains with a total acid content of more than 10.0g/L (as lactic acid) and sensory evaluations of A, etc. and B, etc. were selected from the strains, as shown in the following table.

Strain numbering	Lactic acid content (g/L)	Strain numbering	Lactic acid content (g/L)
				706	21.034	717	12.369
1600	14.576	697	12.345
				767	14.183	750	12.167
1143	13.881	977	11.864
				768	13.335	1595	11.827
815	13.269	1446	11.657
				849	12.886	1757	11.510
823	12.802	1602	11.387
				770	12.763	972	11.330
3887	12.689	769	11.112
				1293	12.671	1576	10.967
1161	12.540	693	10.841
				1621	12.506	982	10.801

Example 2

Bacterial strain re-screening capable of consuming large amount of lactose

The strain preserved in glycerol is activated by MRS + 2% lactose liquid medium and cultured overnight at 37 ℃. And (4) continuously passaging twice, centrifuging the bacterial suspension to remove supernatant, and resuspending bacterial sludge by 5mL of skim milk. Inoculating into 200mL sterilized skim milk according to the volume ratio of 2%, standing and fermenting at 40 deg.C for 24h, and demulsifying to terminate fermentation. The lactose content in the fermented milk is detected by adopting high performance liquid chromatography. Sample pretreatment: accurately weighing 1.00g of sample, adding absolute ethyl alcohol with equal mass, shaking for 30s, centrifuging at 12000rmp for 10min at low temperature, taking a proper amount of supernatant, filtering with a 0.22 mu m filter membrane, and keeping at-20 ℃ for later use. Chromatographic conditions are as follows: liquid chromatograph, Agilent 1200, column, Hypersil NH₂(4.6mm × 300mm, 5 μm), mobile phase, acetonitrile: water 75: 25, flow rate, 1.4mL/min, detector, refractive index detector, elution time, 20min, column temperature: at 30 ℃. The chromatogram peak of the mixed sugar standard is shown in figure 1. And (3) preparing a standard curve: lactose standards were formulated as gradient solutions of 50, 30, 20, 10, 5, 2.5mg/mL and assayed according to the methods and chromatographic conditions described above. The concentration of the standard was used as the abscissa and the peak area was used as the ordinate to plot a standard curve, as shown in fig. 2.

The lactose content in the fermented milk of the lactobacillus delbrueckii subspecies bulgaricus 3887 strain is the lowest. The sensory quality of the strain fermented milk is evaluated as a, etc., and can be described as the fermented milk curd is good, milky white, less whey, and produces a pleasant aroma.

Strain numbering	Lactose content (g/100g)	Strain numbering	Lactose content (g/100g)
				706	2.60	717	2.29
1600	2.12	697	2.09
				767	1.96	750	2.36
1143	2.17	977	2.40
				768	2.36	1595	1.86
815	2.27	1446	2.69
				849	2.32	1757	1.92
823	1.76	1602	2.57
				770	1.86	972	2.42
3887	1.41	769	2.50
				1293	2.32	1576	2.29
1161	2.49	693	1.59
				1621	2.37	982	2.57

Example 3

Microbiological Properties of Lactobacillus delbrueckii subspecies bulgaricus WHH3887

The microbial properties of strain WHH3887 are shown below:

(1) the culture temperature is preferably 37 ℃;

(2) the culture medium is preferably MRS + 2% lactose culture medium;

(3) gram-positive, rod-like, spore-free, flagellum-free (fig. 3A), catalase-negative, oxidase-negative;

(4) the colony morphology is characterized in that: culturing in MRS modified solid culture medium at 37 deg.C for 48-72 hr to obtain semitransparent colony with irregular and jagged edge and less luster (FIG. 3B);

(5) carbohydrate acidogenesis profile (API50 CH):

glycerol	-	Inositol	-	Inulin	-
						Erythritol and its preparation method	-	Mannitol	-	Melezitose	-
D-arabinose	-	Sorbitol	-	Cotton seed candy	-
						L-arabinose	-	alpha-methyl-D-mannoside	-	Starch	-
D-ribose	-	alpha-methyl-D-glucoside	-	Glycogen	-
						D-xylose	-	N-acetyl-glucosamine	+	Xylitol, its preparation method and use	-
L-xylose	-	Amygdalin	-	Gentiobiose	-
						Adone alcohol	-	Arbutin	-	D-turanose	-
beta-methyl-D-xyloside	-	Qiyeling (medicine for treating gynecopathy)	+	D-lyxose	-
						D-galactose	-	Salicin	-	D-tagatose	-
D-glucose	+	Cellobiose	-	D-fucose	-
						D-fructose	+	Maltose	-	L-fucose	-
D-mannose	+	Lactose	+	D-arabinitol	-
						L-sorbose	-	Sucrose	-	L-arabinitol	-
L-rhamnose	-	Trehalose	-	Gluconate	-
						Dulcitol	-	Melibiose	-	2-keto-gluconate	-

(6)16S rDNA gene sequence determination results:

(7) pheS gene sequencing results:

according to the comprehensive analysis of the cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, pheS gene sequence and other data of the strain, the identification result of the strain is Lactobacillus delbrueckii subsp. No. 3 of Xilu No. 1 of Beijing, Chaoyang, and the preservation number is CGMCC NO. 20090.

Example 4

Acid and bile salt resistance experiment

(1) Determination of tolerance ability: after two successive passages of an experimental strain (Lactobacillus delbrueckii subspecies bulgaricus WHH3887) and control strains WHH195 and WHH200 (both Lactobacillus delbrueckii subspecies bulgaricus, respectively identified as control strain A, B), the bacterial suspensions were inoculated at 3% (v/v) into MRS liquid media with pH values of 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0, respectively, and anaerobically cultured at 37 ℃ for 12 h. The absorbance at 600nm of each group of cultures was measured in parallel 3 times. The OD of the culture at pH 6.0 was used as a control. The experimental results show that the acid resistance of the experimental strain 3887 is significantly higher than that of the control strain A and the control strain B. At a pH of 3.0, the relative survival rate of strain WHH3887 was 70.13%, which was 1.59-fold higher than that of control strain A and 2.11-fold higher than that of control strain B. The pH value of normal human gastric juice fluctuates between 1.5 and 4.5, and the strain WHH3887 can maintain a high survival rate under the condition that the pH value is 3 to 4, so that the strain has excellent acid tolerance.

(2) And (3) testing the bile salt resistance: after two successive passages of the experimental strain (Lactobacillus delbrueckii subspecies Bulgaria WHH3887) and the control strain (both the control strain A and the control strain B are Lactobacillus delbrueckii subspecies Bulgaria), the bacterial suspensions were inoculated at 3% (v/v) into MRS liquid medium containing 0%, 0.3%, 0.5% and 1.0% of bovine bile salts, and cultured anaerobically at 37 ℃ for 12 h. The absorbance at 600nm of each group of cultures was measured in parallel 3 times. The OD value of 0% ox-bile-salt culture was used as a control. The results of the experiment show that the relative survival rate of the experimental strain WHH3887 is 68.56% which is 1.21 times that of the control strain A and 1.37 times that of the control strain B when the content of the bile salts in the culture medium is 0.5%. At present, the unified scientific standard of the lactobacillus for tolerating the bile salt is not determined internationally, but experimental data at home and abroad show that if the strain can maintain a certain survival rate under the condition that the content of the bile salt is 0.5%, the strain can be indirectly proved to have higher bile salt tolerance.

Example 5

Experiment of antioxidant ability

(1) Determination of hydrogen peroxide resistance: after two successive passages of an experimental strain (Lactobacillus delbrueckii subspecies bulgaricus WHH3887) and a control strain (both the control strain A and the control strain B are Lactobacillus delbrueckii subspecies bulgaricus), the bacterial suspension is inoculated in MRS liquid culture media with hydrogen peroxide concentrations of 0.4, 0.7 and 1.0mmol/L according to 3% (v/v), and is subjected to anaerobic culture at 37 ℃ for 12 hours. The absorbance of each group of culture at 600nm was measured for 3 times in parallel, and the growth of the cells was observed under hydrogen peroxide conditions of different concentrations. The experimental result shows that the growth of the experimental strain WHH3887 is not obviously influenced along with the increase of the concentration of the hydrogen peroxide, and the growth of the control strain A and the growth of the control strain B are obviously reduced when the concentration of the hydrogen peroxide is 0.7mmol/L, which indicates that the strain WHH3887 is insensitive to the hydrogen peroxide and has good antioxidant activity potential.

(2) Measurement of hydroxyl radical scavenging ability: taking 1mL of brilliant green solution (0.435mmol/L) and 1mL of bacterial suspension, and fully and uniformly mixing; then 2mL of FeSO was added₄Solution (0.5mmol/L) and 1.5mL H₂O₂The solution (3%, w/v) was allowed to stand at room temperature for 20min, and then the absorbance at 624nm of the reaction solution was measured and recorded As As. Other conditions were unchanged, and H was replaced by an equal volume of distilled water₂O₂Measuring the light absorption value, and marking as Ab; the absorbance was measured by replacing the sample with an equal volume of distilled water and recorded as Ac.

Hydroxyl radical clearance (%) - (As-Ac)/(Ab-Ac) × 100, where As is the absorbance of the sample set; ac is the light absorption value of the control group; ab is blank absorbance. The experimental results show that the experimental strain and the control strain both have hydroxyl radical scavenging capacity, and the experimental strain WHH3887 has the strongest scavenging capacity.

(3) DPPH radical scavenging: prepare 0.2mmol/L DPPH solution with absolute ethanol. And (3) fully and uniformly mixing 1mL of DPPH solution and 1mL of bacterial suspension, keeping out of the sun, reacting at room temperature for 30min, centrifuging at 6000r/min for 10min, taking the supernatant, measuring the light absorption value at 517nm, and recording As. Replacing DPPH solution with equal volume of absolute ethyl alcohol to measure the light absorption value, and marking as Ab; the absorbance was measured by replacing the sample with an equal volume of distilled water and recorded as Ac. DPPH radical clearance (%) [1- (As-Ab)/Ac ] × 100, where As is the sample set absorbance; ac is the light absorption value of the control group; ab is blank absorbance. The experimental results show that the experimental strain and the control strain both have certain DPPH free radical scavenging capacity, and the experimental strain WHH3887 has the strongest scavenging capacity.

Strain numbering	Hydroxyl radical scavenging rate (%)	DPPH radical scavenging ratio (%)
			WHH3887	22.565±0.07	15.233±0.08
WHH195	12.899±0.08	14.778±0.11
			WHH200	12.433±0.10	14.922±0.12

Example 6

Preparation of lactobacillus delbrueckii subspecies bulgaricus WHH3887 direct vat set starter

Single colonies of solid plates were picked up in MRS + 2% lactose liquid medium and cultured overnight at 37 ℃ under static conditions. Inoculating the bacterial suspension into an OPL culture medium (fermentation optimization culture medium) according to the volume ratio of 3 percent for continuous passage twice for amplification culture, inoculating into a 10L fermentation tank for anaerobic fermentation, centrifuging fermentation liquor after fermentation is finished, removing supernatant, uniformly mixing collected bacterial sludge and a protective agent according to a certain proportion, and freeze-drying the mixture in a freeze dryer to obtain WHH3887 freeze-dried bacterial powder which is a direct vat set starter and can be directly used for preparing low-lactose or zero-lactose fermented milk.

Example 7

Application of lactobacillus delbrueckii subspecies bulgaricus WHH3887 in low-lactose or zero-lactose fermented dairy products

Mixing lyophilized powder of Lactobacillus delbrueckii subsp bulgaricus 3887 with lyophilized powder of Streptococcus thermophilus 445 and lactococcus lactis 2353 at a ratio of 1: 1 to obtain direct vat set starter, wherein the initial inoculation amount is 5.0 × 10⁶Inoculating CFU/mL into reconstituted milk, standing at 40 deg.C for 10 hr and 24 hr, demulsifying to stop fermentation, and after-ripening at 4 deg.C for 2-4 hr. The pH change was monitored during the fermentation, the fermented milk was evaluated organoleptically after the end of the fermentation, and the residual lactose content of the fermented milk was determined at two time points, 10h and 24h of the fermentation, according to the method described in example 2.

The strain WHH3887 and each strain can perform synergistic fermentation. Sensory evaluation results show that the fermented milk is thick and stringy, has fine and smooth mouthfeel and is rich in milk fat aroma. The pH value tracking result shows that the acid production capacity of the strain WHH3887 is good, the pH value is 4.51 after 8 hours of fermentation, the pH value can reach 4.52 after 4.5 hours of fermentation after the strain WHH3887 is compounded with the strain 445 and the strain 2353, and the pH value is not obviously changed after the end point of the fermentation (as shown in figure 4). The detection result of the lactose content shows that the strain WHH3887 is fermented for 10 hours by single bacterium, the residual lactose content in the fermented milk is 1.89g/100g, the fermentation is continued to 24 hours, and the residual lactose content is 0.32g/100 g; the strain WHH3887 is compounded with the other two lactic acid bacteria and then fermented for 10 hours, the residual amount of lactose in the fermented milk is 1.51g/100g, the fermentation is continued for 24 hours, the residual amount of lactose is 0.22g/100g, and the content reaches the alleged requirement (less than or equal to 2.0g/100g) of low lactose and the alleged requirement (less than or equal to 0.5g/100g) of zero lactose in GB28050-2011 food safety national standard-prepackaged food nutrition label convention).

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Hangzhou baby Haha science Co Ltd

<120> Lactobacillus delbrueckii strain capable of consuming a large amount of lactose and having acid resistance, cholate resistance and oxidation resistance and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

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<212> DNA/RNA

<213> Lactobacillus delbrueckii subsp. Bulgaricus WHH3887)

<400> 1

tgcaagtcga gcgagctcaa ttcaaagatt ccttcggggt gatttgttgg acgctagcgg 60

cggatgggtg agtaacacgt gggcaatctg ccctaaagac tgggatacca cttggaaaca 120

ggtgctaata ccggataaca acatgaatcg catgattcaa gtttgaaagg cggcgcaagc 180

tgtcacttta ggatgagccc gcggcgcatt agctagttgg tggggtaaag gcctaccaag 240

gcaatgatgc gtagccgagt tgagagactg atcggccaca ttgggactga gacacggccc 300

aaactcctac gggaggcagc agtagggaat cttccacaat ggacgcaagt ctgatggagc 360

aacgcccgtg agtgaagaag gttttcggat cgtaaagctc tgttgttggt ggaagaagga 420

tagaggcagt aactggttct ttatttgacg gtaatcaacc agaaagtcac ggctaactac 480

gtgccagcag ccgcggtaaa aggatagagg cagtaactgg tctttatttt gacggtaatc 540

aaccagaaag tcacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 600

ttgtccggat ttattgggcg taaagcgagc gcaggcggaa tgataagtct gatgttgaaa 660

gcccacggct caaccgtgga actgcatcgg aaactgtcat tcttgagtgc agaagaggag 720

agtggaattc catgtgtagc ggtggaatgc gtagatatat ggaagaacac cagtggcaag 780

gcggctctct ggtctgcaac tgacgctgag gctcgaaagc atgggtagcg aacaggatta 840

gataccctgg tagtccatgc cgtaaacgat gagcgctagg tgttggggac tttccggtcc 900

tcagtgccgc agcaaacgca ttaagcgctc cgcctgggga gtacgccgca aggttgaaac 960

tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 1020

gcgaagaacc ttaccaggtc ttgacatcct gtgctacacc tagagatagg tggttccctt 1080

cggggacgca gagacaggtg gtgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 1140

taagtcccgc aacgagcgca acccttgtcc ctttagttgc catcattaag ttgggcactc 1200

taaagagact gccggtgaca aaccggagga aggtggggga tgacgtcaag tcatcatgcc 1260

ccttatgacc tgggctacac acgtgctaca atgggcagta caacgagaag cgaacccgcg 1320

agggtaagcg gatctcttaa agctgttctc agttcggact gcaggctgca actcgcctgc 1380

acgaagctgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccgggc 1440

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ataaagggga cctcaagatg atctccccag gcaaggttta cagaagagac gatgacgacg 120

ccacccactc ccaccagttc atgcagatgg aagggctggt tgtcggcaag aacatctcct 180

tgagtgacct taaggggacc ttggaactgg tggccaagca cgaattcggc caggaccggg 240

aaacccgctt gcggccaagc tacttcccat ttactgaacc atcagttgaa atggacgttt 300

cttgctttga atgcggcggc aagggctgcg cgatctgcaa gaacaccggc ctggatcgaa 360

gttctgggtg ccgggatcgt tcacccgaat gttttgtctg ccgccggcat tgacccaagc 420

gtctactctg gctttgc 437

Claims

1. A lactobacillus delbrueckii strain capable of consuming a large amount of lactose and having acid resistance, cholate resistance and oxidation resistance is characterized in that: the lactobacillus delbrueckii is named as WHH3887 and has been preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 15.6.2020, the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaoyang, and the preservation number is CGMCC No. 20090; the microorganism classification is named as Lactobacillus delbrueckii subspecies bulgaricusLactobacillus delbrueckii subsp.bulgaricus。

2. A mutant of Lactobacillus delbrueckii capable of consuming a large amount of lactose and having acid resistance, bile salt resistance and oxidation resistance, wherein the mutant is obtained by subjecting the Lactobacillus delbrueckii of claim 1 to mutagenesis, acclimation, genetic recombination or natural mutation.

3. The mutant according to claim 2, which has a nucleotide sequence having at least 90% or more homology with lactobacillus delbrueckii as defined in claim 1, and which has at least 90% or more of lactose resolving power, acid resistance, bile salt resistance and oxidation resistance, respectively, with lactobacillus delbrueckii as defined in claim 1.

4. The mutant according to claim 3, wherein the mutant has a nucleotide sequence at least 95% homologous to the Lactobacillus delbrueckii strain of claim 1, and the mutant has at least 95% or more of lactose degradation ability, acid resistance, bile salt resistance and oxidation resistance, respectively, to the Lactobacillus delbrueckii strain of claim 1.

5. A bacterial culture comprising the Lactobacillus delbrueckii strain according to claim 1 or comprising the mutant according to any of claims 2 to 5.

6. Use of the Lactobacillus delbrueckii strain according to claim 1, or the mutant according to any of claims 2 to 4, or the cell culture according to claim 5, for the preparation of a lactobacillus direct vat set starter.

7. The application of the lactobacillus delbrueckii, the streptococcus thermophilus 445 and the lactococcus lactis 2353 in preparation of the lactobacillus direct vat set starter after being compounded in claim 1.

8. The use of claim 7, wherein the ratio of Lactobacillus delbrueckii to Streptococcus thermophilus 445 and lactococcus lactis 2353 is (0.8-1.2): (0.8-1.2).

9. Use according to any one of claims 6 to 8, wherein the lactic acid bacteria ready-to-use starter is used for the preparation of low-lactose fermented milk, zero-lactose fermented milk, fermented dairy milk, low-lactose lactic acid bacteria drink, zero-lactose lactic acid bacteria drink and regular lactic acid bacteria drink.

10. The use according to claim 6 or 7, wherein the lactic acid bacteria direct vat set is used for preparing food, beverage, health food and medicine for improving lactose malabsorption, lactose indigestion and lactose intolerance.