CN110129243B

CN110129243B - Soy yogurt suitable strains, inoculants and their applications

Info

Publication number: CN110129243B
Application number: CN201910534345.8A
Authority: CN
Inventors: 方祥; 余建英; 郑勇; 王洁; 廖振林
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-06-20
Filing date: 2019-06-20
Publication date: 2021-02-23
Anticipated expiration: 2039-06-20
Also published as: CN110129243A

Abstract

The invention relates to the technical field of microorganisms, in particular to a bean yogurt adaptive strain, a microbial inoculum and application thereof. The soy yogurt adaptive strain is selected from any one or more of the following strains: 1) lactobacillus rhamnosus SCB0119, the preservation number is: CGMCC 17618; 2) lactococcus lactis SCB0469 with the deposit number: CGMCC 17619; 3) lactobacillus helveticus SCB0641 with a collection number of: CGMCC 17620; all three strains are preserved in China center for culture Collection of microorganisms for 24 days at 4 months in 2019. The microbial inoculum is obtained by reasonable compatibility, and the mixed soybean milk is fermented to obtain the soybean yogurt with good curd effect and good flavor, and the soybean isoflavone can be biologically converted to improve the content of free soybean isoflavone aglycone.

Description

Soybean yogurt adaptive strain, microbial inoculum and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a bean yogurt adaptive strain, a microbial inoculum and application thereof.

Background

The traditional yoghourt is an animal fermented food which is obtained by taking fresh milk or reconstituted milk as a raw material and carrying out processing by process technologies such as blending, homogenizing, sterilizing, cooling, inoculating a leavening agent and the like and microbial fermentation. The bean yoghourt is a plant (or animal) fermented food which is obtained by taking fresh soybean milk or recovered soybean milk as a raw material, adding or not adding fresh milk or recovered milk, processing by the process technologies of blending, homogenizing, sterilizing, cooling, inoculating a leavening agent and the like, and fermenting by microorganisms.

After the fresh milk or the reconstituted milk is added in the preparation of the bean yoghourt, compared with the traditional yoghourt, the soybean yoghourt combines the advantages of the milk and the soybean and simultaneously makes up the defects of the milk and the soybean, and has more comprehensive and rich protein types, amino acid compositions, fat types and vitamin types and more balanced nutrition. The addition of the milk makes up the defects of poor curding capability, strong beany flavor and poor taste and flavor when the soybean milk is simply fermented, and the existence of the soybean milk makes up the defect that the traditional yoghurt lacks dietary fibers, and particularly brings the natural active substances of soybean isoflavone to the soybean yoghurt.

The soybean isoflavone is a natural active substance existing in soybeans, has various active effects of protecting cardiovascular system, preventing atherosclerosis, preventing osteoporosis, relieving female climacteric symptoms, resisting tumor and oxidation, protecting nerves, regulating immunity, protecting liver and the like, and has excellent application prospect and market prospect. At present, some organizations in Japan propose that the upper limit of the intake of soybean isoflavone in daily diet is 70-75 mg/d, and the upper limit of the intake of soybean isoflavone added outside diet is 30 mg/d. In European Union, the recommended intake of soybean isoflavone is 40-100 mg/d according to EC 1924/2006.

Under natural conditions, the content of soybean isoflavone in soybeans is about 0.1-0.5%, 12 isomers are generally considered, including 9 bound glycosides mainly comprising daidzin, genistin and the like and 3 free-type aglycones comprising daidzein, genistein and glycitein, wherein the bound glycoside accounts for 97-98%, and the free-type aglycone only accounts for 2-3%. Change and Nair (1995) found that soybean isoflavone glycoside is difficult to be absorbed and utilized by human body, compared with soybean isoflavone aglycone which is easier to be absorbed and utilized by human body. Xu et al (1995) and Wei et al (1996) have suggested that soybean isoflavone aglycones have higher biological activity than soybean isoflavone glycosides, and that soybean isoflavones have multiple active effects when present in the aglycone form. The soybean isoflavone glycoside can be hydrolyzed into soybean isoflavone aglycone by beta-glucosidase secreted by part of intestinal microorganisms, but the composition of intestinal flora varies from person to person, so that the conditions of synthesizing and secreting the beta-glucosidase and converting the soybean isoflavone in a human body are different, and the amount of the soybean isoflavone aglycone which is finally converted is limited. In conclusion, the biological conversion of the soybean isoflavone can effectively improve the biological activity and the bioavailability of the soybean isoflavone in the organism, and has very important significance.

According to data records of soybean germplasm resource databases of the institute of crop science of the academy of agricultural sciences of China and soybean germplasm resource databases of the department of agriculture of the United states, the content of protein in soybeans is 30.8-57.9%, the soybean protein is high-quality protein like milk protein, and the soybean protein mainly comprises albumin (accounting for about 10%) and globulin (accounting for about 90%). According to immunological analysis, globulin contains 40% of glycinin and 30% of beta-conglycinin, which are main antigen proteins in soybean and are easy to cause adverse reactions such as allergy and the like. However, the two antigen proteins have stronger heat stability, and the heat treatment at the temperature of 100 ℃ has little influence on the antigen activity. Therefore, researchers have turned to methods for removing antigenic proteins, and have improved the nutritional properties of soy products by reducing their antigenic activity through fermentation with microorganisms such as lactic acid bacteria.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a soy yogurt adaptive strain, which can biologically convert soy isoflavone, thereby improving the content of aglycon soy isoflavone components and improving the biological activity and bioavailability in a body.

The second purpose of the invention is to provide a bean yoghourt adaptive microbial inoculum, which can be used for bean yoghourt adaptive fermentation after the bacterial strain is scientifically matched, reduce the content of antigen protein in the bean yoghourt, and release and improve the nutrition of the bean yoghourt.

The third purpose of the invention is to provide the application of the bean yoghourt adaptive microbial inoculum in the preparation of the bean yoghourt, and the bean yoghourt prepared by using the bean yoghourt adaptive microbial inoculum can obtain good curd effect and improve the flavor of the bean yoghourt.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a soy yogurt-compatible strain selected from any one or more of the following strains:

a) lactococcus lactis SCB0469, Lactococcus lactis, deposited in the China general microbiological culture Collection center, with the deposit numbers: CGMCC 17619; the preservation time is as follows: 24 months 4 in 2019;

b) lactobacillus rhamnosus SCB0119, Lactobacillus rhamnosus, preserve in China center for culture of microorganisms, the preserving number is: CGMCC 17618; the preservation time is as follows: 24 months 4 in 2019;

c) lactobacillus helveticus SCB0641, Lactobacillus helveticus, is preserved in China center for culture of microorganisms with the preservation number: CGMCC 17620; the preservation time is as follows: 24/4/2019.

The morphological description characteristics of each strain are respectively as follows: a colony morphology chart obtained after lactobacillus rhamnosus SCB0119 strain is inoculated on an MRS solid culture medium and cultured for 72 hours at the temperature of 37 ℃. The colony diameter is 1.48-2.75mm, and the colony is round, white, glossy, opaque, moist, low around the middle bulge, and smooth and complete edge.

A colony morphology obtained after the lactococcus lactis strain SCB0469 was inoculated on an MRS solid medium and cultured at a temperature of 30 ℃ for 72 hours. The colony diameter is 1.42-2.90mm, and the colony is round, white, glossy, slightly transparent, moist, slightly convex in the middle, low in periphery, smooth and complete in edge.

A colony morphology obtained after lactobacillus helveticus SCB0641 strain was inoculated on MRS solid medium and cultured at 37 ℃ for 72 hours. The colony diameter is 1.35-2.20mm, and the colony is round, white, glossy, slightly transparent, slightly moist, low in middle protrusion, smooth and complete in edge.

The 16SrDNA sequence of Lactobacillus rhamnosus (Lactobacillus rhamnosus) SCB0119 has 1380 nucleotides, and the sequence is shown as SEQ ID NO: 1 is shown.

The 16SrDNA sequence of Lactococcus lactis (Lactococcus lactis) SCB0469 has 1419 nucleotides, and the sequence is shown as SEQ ID NO: 2, respectively.

The 16SrDNA sequence of Lactobacillus helveticus (Lactobacillus helveticus) SCB0641 has 1409 nucleotides, and the sequence is shown as SEQ ID NO: 3, respectively.

The three strains are all preserved in the China general microbiological culture Collection center, and the preservation addresses are as follows: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The lactococcus lactis SCB0469 has the main effects of serving as a soy yogurt flavor strain and improving the flavor of soy yogurt, the lactobacillus rhamnosus SCB0119 has the main effects of removing antigen protein in soy milk and converting soy isoflavone, and the lactobacillus helveticus SCB0641 has the main effects of converting soy isoflavone and improving the content of aglycone soy isoflavone in the soy yogurt.

The bean yoghurt adaptive strain is a safe edible strain which has any one or more of the following effects by fermenting pure bean milk (or mixed bean milk): (1) can produce pleasant flavor, (2) can effectively remove anti-nutritional factors, and (3) can biologically convert the soybean isoflavone to improve the content of the aglycone soybean isoflavone so as to improve the biological activity and the bioavailability of the aglycone soybean isoflavone.

According to the strain provided by the invention, the strain number of the lactobacillus rhamnosus is SCB0119, the strain number of the lactococcus lactis is SCB0469, and the strain number of the lactobacillus helveticus is SCB 0641.

The lactobacillus rhamnosus SCB0119 provided by the invention is separated from Sichuan pickle and is obtained by dilution and pouring plate separation.

The lactococcus lactis SCB0469 provided by the invention is separated from Russian kefir and is obtained by dilution and pouring on a plate.

The Lactobacillus helveticus SCB0641 provided by the invention is separated from Tibetan spiritual mushroom by a dilution and pouring plate method.

The invention claims the bean yoghourt adaptive strain with the preservation number and the mutant strain which has mutation in a moderate range and still has strong bean yoghourt adaptive capability.

In the practical application process, the bean yoghourt adaptive strain can be expanded and cultured to form a composition, namely a bean yoghourt adaptive starter (or a bean yoghourt adaptive microbial inoculum) which is in the form of a microbial inoculum so as to expand the application range of the bean yoghourt adaptive bacterial strain in consideration of the possible transportation and other reasons.

The bean yoghourt adaptive starter is a bean yoghourt direct vat set type freeze-dried starter prepared by 2 or more lactic acid bacteria strains which are prepared by scientifically matching bean yoghourt adaptive strains, can generate pleasant flavor, can biologically convert soybean isoflavone to improve the content of aglycon type soybean isoflavone so as to improve the biological activity and the bioavailability and/or can effectively remove anti-nutritional factors and improve the nutritional efficacy of the bean yoghourt.

The invention also provides a microbial inoculum containing the strain.

In some embodiments of the present invention, the microbial inoculum may further include any one or more of streptococcus thermophilus, lactobacillus plantarum, lactobacillus casei and lactobacillus paracasei.

In some embodiments of the present invention, the microbial inoculum preferably comprises at least lactococcus lactis SCB 0469.

The strain can be the existing strain, and can improve the fermentation effect and the like by being matched with the strain obtained by separation.

Specifically, the streptococcus thermophilus, the lactobacillus plantarum, the lactobacillus casei and the lactobacillus paracasei can be used as the bacteria for fermenting the common yoghourt.

In some specific embodiments of the invention, in the microbial inoculum, the ratio of the lactobacillus rhamnosus SCB0119 to the lactobacillus lactis SCB0469 to the lactobacillus helveticus SCB0641 is (0-1.22 × 10) in terms of the number of viable bacteria¹⁰)﹕ (1.0×10⁶～3.60×10¹⁰)﹕(0～8.55×10¹⁰). Preferably, the ratio of the three bacteria in the microbial inoculum is (1X 10) in terms of viable count⁷～8×10⁹)﹕(1×10⁷～8×10⁹)﹕(1×10⁷～8×10⁹) (ii) a More preferably, the ratio of the three bacteria in the microbial inoculum is (6.5X 10) in terms of viable count⁷～6×10⁹)﹕(1.2×10⁷～5.5×10⁹)﹕(1.8×10⁷～ 4×10⁹)。

In some specific embodiments of the invention, in the microbial inoculum, the ratio of the lactobacillus rhamnosus SCB0119, the lactobacillus lactis SCB0469, the lactobacillus helveticus SCB0641 and the streptococcus thermophilus is (0-1.22 × 10) in terms of the number of viable bacteria¹⁰) ﹕(1.0×10⁶～3.60×10¹⁰)﹕(0～8.55×10¹⁰)﹕(0～4.54×10⁹). Preferably, the ratio of the four bacteria in the microbial inoculum is (1X 10) in terms of viable count⁷～8×10⁹)﹕(1×10⁷～8×10⁹)﹕(1×10⁷～8×10⁹) ﹕(1×10⁶～4×10⁹) (ii) a More preferably, the ratio of the four bacteria in the microbial inoculum is (6.5X 10) in terms of viable count⁷～6×10⁹) ﹕(1.2×10⁷～5.5×10⁹)﹕(1.8×10⁷～4×10⁹)﹕(3×10⁶～3×10⁹)。

The specific implementation mode of the invention provides several scientific compatibility modes, which are as follows:

the microbial inoculum comprises Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, lactococcus lactis SCB0469 and Lactobacillus paracasei. The ratio of Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, lactococcus lactis SCB0469 and Lactobacillus paracasei is preferably (6.5X 10) in terms of viable count⁷～3.0×10⁹)﹕(1.5×10⁸～5.0×10⁹) ﹕(1.5×10⁸～3.0×10⁹)﹕(4.0×10⁸～7.5×10⁹). More preferably, the microbial inoculum further comprises streptococcus thermophilus. Further preferably, the microbial inoculum also comprises lactobacillus plantarum and/or lactobacillus casei.

When the microbial inoculum is adopted, in some specific embodiments, the ratio of the lactobacillus rhamnosus SCB0119, the lactobacillus helveticus SCB0641, the lactococcus lactis SCB0469, the lactobacillus paracasei and the streptococcus thermophilus in the microbial inoculum is (6.5 multiplied by 10)⁷～2.5×10⁹)﹕(4.0×10⁸～4.0×10⁹)﹕(1.5×10⁸～2.5×10⁹)﹕ (4.5×10⁸～7.0×10⁹)﹕(2.0×10⁷～3.0×10⁹). Preferably, in some embodiments of the present invention, the ratio of Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, lactococcus lactis SCB0469, Lactobacillus paracasei, and Streptococcus thermophilus is (9.0 × 10) based on the number of viable bacteria⁷～5.0×10⁸)﹕(4.0×10⁸～8.0×10⁸)﹕ (1.5×10⁸～5.0×10⁸)﹕(1.0×10⁹～7.0×10⁹)﹕(2.0×10⁷～6.0×10⁷)。

When the microbial inoculum is adopted, in some specific embodiments, the ratio of the lactobacillus rhamnosus SCB0119, the lactobacillus helveticus SCB0641, the lactococcus lactis SCB0469, the lactobacillus paracasei, the streptococcus thermophilus, the lactobacillus plantarum and the lactobacillus casei in the microbial inoculum is (6.5 multiplied by 10)⁷～2.5×10⁹)﹕(4.0×10⁸～4.0×10⁹)﹕ (1.5×10⁸～2.5×10⁹)﹕(4.5×10⁸～7.0×10⁹)﹕(2.0×10⁷～3.0×10⁹)﹕(3.0×10⁸～ 5.0×10⁹)﹕(5.5×10⁸～5.5×10⁹). Preferably, in some embodiments of the present invention, the ratio of Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, lactococcus lactis SCB0469, Lactobacillus paracasei, Streptococcus thermophilus, Lactobacillus plantarum, and Lactobacillus casei is (3.5 × 10) based on the number of viable bacteria⁸～2.5×10⁹)﹕(4.0×10⁸～ 4.0×10⁹)﹕(1.5×10⁸～2.5×10⁹)﹕(4.5×10⁸～5.5×10⁹)﹕(1.0×10⁸～3.0×10⁹) ﹕(3.0×10⁸～5.0×10⁹)﹕(5.5×10⁸～5.5×10⁹)。

The microbial inoculum comprises lactococcus lactis SCB0469 and Lactobacillus helveticus SCB 0641. The ratio of lactococcus lactis SCB0469 to Lactobacillus helveticus SCB0641 is preferably (1.2X 10) in terms of the number of viable bacteria⁷～2.5×10⁹)﹕(1.8×10⁷～ 4.0×10⁹). More preferably, the microbial inoculum further comprises streptococcus thermophilus and/or lactobacillus paracasei.

When the microbial inoculum is adopted, in some specific embodiments, the ratio of the lactococcus lactis SCB0469, the Lactobacillus helveticus SCB0641 and the Streptococcus thermophilus is (1 multiplied by 10) in terms of the number of the viable bacteria in the microbial inoculum⁷～2.5×10⁹)﹕(1.5×10⁷～ 4.0×10⁹)﹕(3×10⁶～3×10⁹). Preferably, in the present inventionIn some embodiments, the ratio of the viable bacteria count of the lactococcus lactis SCB0469, the Lactobacillus helveticus SCB0641 and the Streptococcus thermophilus is (1.2 × 10)⁷～1.5×10⁷) ﹕(1.8×10⁷～2.5×10⁷)﹕(3×10⁶～8×10⁶). Or the ratio of Lactobacillus helveticus SCB0641, Lactobacillus paracasei and lactococcus lactis SCB0469 in terms of viable count in the microbial inoculum is (3.0 × 10)⁸～5.0×10⁹)： (4.0×10⁸～5.5×10⁹)﹕(1.5×10⁸～2.5×10⁹). More preferably, the ratio of the lactobacillus helveticus SCB0641, the lactobacillus paracasei SCB0469 and the streptococcus thermophilus in the microbial inoculum is (4.0 × 10) in terms of the number of the viable bacteria⁸～4.0×10⁹)：(4.5×10⁸～5.5×10⁹)﹕(1.5×10⁸～2.5×10⁹)﹕(1.0×10⁸～ 3.0×10⁹). Preferably, in some embodiments of the present invention, the ratio of Lactobacillus helveticus SCB0641, Lactobacillus paracasei, lactococcus lactis SCB0469, Streptococcus thermophilus (4.0X 10) is calculated by the number of viable bacteria⁸～6.0×10⁸)： (4.5×10⁸～9.5×10⁸)﹕(6×10⁸～1.0×10⁹)﹕(5.0×10⁸～1.5×10⁹)。

The microbial inoculum comprises Lactobacillus rhamnosus SCB0119 and Lactobacillus lactis SCB 0469. In the microbial inoculum, the ratio of Lactobacillus rhamnosus SCB0119 to lactococcus lactis SCB0469 is preferably (6.5 multiplied by 10) in terms of the number of viable bacteria⁷～6×10⁹) ﹕(1.5×10⁸～3.5×10⁹). More preferably, the microbial inoculum further comprises streptococcus thermophilus.

When the microbial inoculum is adopted, in some specific embodiments, the ratio of the lactobacillus rhamnosus SCB0119, the lactococcus lactis SCB0469 and the streptococcus thermophilus is (6.5 multiplied by 10) in terms of the number of the viable bacteria in the microbial inoculum⁷～6×10⁹)﹕(1.5×10⁸～ 3.5×10⁹)﹕(2×10⁷～3×10⁹). Preferably, in some embodiments of the present invention, the ratio of Lactobacillus rhamnosus SCB0119, lactococcus lactis SCB0469 and Streptococcus thermophilus, based on the number of viable bacteria, is: (1×10⁹～6×10⁹)﹕ (1×10⁹～3.5×10⁹)﹕(8×10⁷～8×10⁸)。

The microbial inoculum comprises lactobacillus casei and lactococcus lactis SCB 0469. In the microbial inoculum, the ratio of lactobacillus casei to lactococcus lactis SCB0469 is preferably (4 × 10) in terms of viable count⁸～8×10⁹)﹕(1×10⁸～2.5×10⁹). More preferably, the microbial inoculum further comprises streptococcus thermophilus.

When the microbial inoculum is adopted, in some specific embodiments, the ratio of the lactobacillus casei, the lactococcus lactis SCB0469 and the streptococcus thermophilus in the microbial inoculum is (5.5 multiplied by 10) in terms of viable count⁸～5.5×10⁹)﹕(1.5×10⁸～2.5×10⁹) ﹕(4×10⁷～3×10⁹). Preferably, in some embodiments of the present invention, the ratio of Lactobacillus casei, lactococcus lactis SCB0469 and Streptococcus thermophilus is (1.0X 10) based on the number of viable bacteria⁹～5.5×10⁹)﹕(9×10⁸～2.5×10⁹) ﹕(4×10⁷～8×10⁷)。

The microbial inoculum comprises lactobacillus paracasei and lactococcus lactis SCB 0469. In the microbial inoculum, the ratio of lactobacillus paracasei to lactococcus lactis SCB0469 is (3 multiplied by 10) in terms of the number of viable bacteria⁸～7.0×10⁹)﹕(1.5×10⁸～2.5×10⁹). More preferably, the microbial inoculum further comprises streptococcus thermophilus.

When the microbial inoculum is adopted, in some specific embodiments, the ratio of the lactobacillus paracasei, the lactococcus lactis SCB0469 and the streptococcus thermophilus in the microbial inoculum is (4.5 multiplied by 10)⁸～7.0×10⁹)﹕(1.5×10⁸～ 2.5×10⁹)﹕(2.0×10⁷～3.0×10⁹). Preferably, in some embodiments of the present invention, the ratio of Lactobacillus paracasei, lactococcus lactis SCB0469 and Streptococcus thermophilus is (1X 10) based on the number of viable bacteria⁹～7.0×10⁹) ﹕(2×10⁸～6×10⁸)﹕(1×10⁸～8×10⁸)。

The microbial inoculum comprises lactobacillus plantarum and lactococcus lactis SCB 0469. The ratio of Lactobacillus plantarum to lactococcus lactis SCB0469 in terms of viable count in the microbial inoculum is preferably (3 × 10)⁸～8×10⁹)﹕(1.5×10⁸～8.0×10⁹). More preferably, the microbial inoculum further comprises streptococcus thermophilus.

When the microbial inoculum is adopted, in some specific embodiments, the ratio of the lactobacillus plantarum to the lactococcus lactis SCB0469 to the streptococcus thermophilus is (3 x 10) in terms of the number of viable bacteria in the microbial inoculum⁸～5×10⁹)﹕(1.5×10⁸～5.5×10⁹)﹕ (1.0×10⁸～3.0×10⁹). Preferably, in some embodiments of the present invention, the ratio of Lactobacillus plantarum, lactococcus lactis SCB0469, and Streptococcus thermophilus is (3X 10) in terms of viable count⁸～6×10⁸)﹕(1×10⁹～5.5×10⁹) ﹕(1.0×10⁸～3.0×10⁹)。

In different embodiments, the bacterial agent can be 0, 1 × 10 and 1 × 10 in terms of the number of active bacteria of lactobacillus rhamnosus SCB0119⁷cfu/L、2×10⁷cfu/L、3×10⁷cfu/L、4×10⁷cfu/L、5×10⁷cfu/L、6×10⁷cfu/L、 7×10⁷cfu/L、8×10⁷cfu/L、9×10⁷cfu/L、1×10⁸cfu/L、2×10⁸cfu/L、3×10⁸cfu/L、 4×10⁸cfu/L、5×10⁸cfu/L、6×10⁸cfu/L、7×10⁸cfu/L、8×10⁸cfu/L、9×10⁸cfu/L、 1×10⁹cfu/L、2×10⁹cfu/L、3×10⁹cfu/L、4×10⁹cfu/L、5×10⁹cfu/L、6×10⁹cfu/L、 7×10⁹cfu/L、8×10⁹cfu/L、9×10⁹cfu/L、1×10¹⁰cfu/L、1.22×10¹⁰cfu/L, etc.;

lactococcus lactis SCB0469 may be: 1.0X 10⁶cfu/L、2×10⁶cfu/L、3×10⁶cfu/L、 4×10⁶cfu/L、5×10⁶cfu/L、6×10⁶cfu/L、7×10⁶cfu/L、8×10⁶cfu/L、9×10⁶cfu/L、 1×10⁷cfu/L、2×10⁷cfu/L、3×10⁷cfu/L、4×10⁷cfu/L、5×10⁷cfu/L、6×10⁷cfu/L、 7×10⁷cfu/L、8×10⁷cfu/L、9×10⁷cfu/L、1×10⁸cfu/L、2×10⁸cfu/L、3×10⁸cfu/L、 4×10⁸cfu/L、5×10⁸cfu/L、6×10⁸cfu/L、7×10⁸cfu/L、8×10⁸cfu/L、9×10⁸cfu/L、 1×10⁹cfu/L、2×10⁹cfu/L、3×10⁹cfu/L、4×10⁹cfu/L、5×10⁹cfu/L、6×10⁹cfu/L、 7×10⁹cfu/L、8×10⁹cfu/L、9×10⁹cfu/L、1×10¹⁰cfu/L、2×10¹⁰cfu/L、3×10¹⁰cfu/L、 3.6×10¹⁰cfu/L, etc.;

lactobacillus helveticus SCB0641 can be 0, 1 × 10⁷cfu/L、2×10⁷cfu/L、3×10⁷cfu/L、 4×10⁷cfu/L、5×10⁷cfu/L、6×10⁷cfu/L、7×10⁷cfu/L、8×10⁷cfu/L、9×10⁷cfu/L、 1×10⁸cfu/L、2×10⁸cfu/L、3×10⁸cfu/L、4×10⁸cfu/L、5×10⁸cfu/L、6×10⁸cfu/L、 7×10⁸cfu/L、8×10⁸cfu/L、9×10⁸cfu/L、1×10⁹cfu/L、2×10⁹cfu/L、3×10⁹cfu/L、 4×10⁹cfu/L、5×10⁹cfu/L、6×10⁹cfu/L、7×10⁹cfu/L、8×10⁹cfu/L、9×10⁹cfu/L、 1×10¹⁰cfu/L、2×10¹⁰cfu/L、3×10¹⁰cfu/L、4×10¹⁰cfu/L、5×10¹⁰cfu/L、6×10¹⁰cfu/L、7×10¹⁰cfu/L、8×10¹⁰cfu/L、8.55×10¹⁰cfu/L, etc.;

the Streptococcus thermophilus can be 0, 1 × 10⁶cfu/L、2×10⁶cfu/L、3×10⁶cfu/L、4×10⁶cfu/L、 5×10⁶cfu/L、6×10⁶cfu/L、7×10⁶cfu/L、8×10⁶cfu/L、9×10⁶cfu/L、1×10⁷cfu/L、 2×10⁷cfu/L、3×10⁷cfu/L、4×10⁷cfu/L、5×10⁷cfu/L、6×10⁷cfu/L、7×10⁷cfu/L、 8×10⁷cfu/L、9×10⁷cfu/L、1×10⁸cfu/L、2×10⁸cfu/L、3×10⁸cfu/L、4×10⁸cfu/L、 5×10⁸cfu/L、6×10⁸cfu/L、7×10⁸cfu/L、8×10⁸cfu/L、9×10⁸cfu/L、1×10⁹cfu/L、 2×10⁹cfu/L、3×10⁹cfu/L、4×10⁹cfu/L、4.54×10⁹cfu/L, etc.;

the lactobacillus paracasei can be 0, 4 × 10⁸cfu/L、5×10⁸cfu/L、6×10⁸cfu/L、7×10⁸cfu/L、 8×10⁸cfu/L、9×10⁸cfu/L、1×10⁹cfu/L、2×10⁹cfu/L、3×10⁹cfu/L、4×10⁹cfu/L、 5×10⁹cfu/L、6×10⁹cfu/L、7×10⁹cfu/L、7.5×10⁹cfu/L, etc.;

the Lactobacillus plantarum can be 0, 3 × 10⁸cfu/L、4×10⁸cfu/L、5×10⁸cfu/L、6×10⁸cfu/L、 7×10⁸cfu/L、8×10⁸cfu/L、9×10⁸cfu/L、1×10⁹cfu/L、2×10⁹cfu/L、3×10⁹cfu/L、 4×10⁹cfu/L、5×10⁹cfu/L, etc.;

the Lactobacillus casei can be 0, 5.5 × 10⁸cfu/L、6×10⁸cfu/L、7×10⁸cfu/L、8×10⁸cfu/L、 9×10⁸cfu/L、1×10⁹cfu/L、2×10⁹cfu/L、3×10⁹cfu/L、4×10⁹cfu/L、5×10⁹cfu/L、 5.5×10⁹cfu/L, and so on.

The microbial inoculum is obtained by scientific compatibility of the strains, can be used for fermenting and mixing the soybean milk, improves the curd effect and the flavor of the obtained soybean yogurt, can improve the conversion rate of the soybean isoflavone in the soybean yogurt, has high content of free soybean isoflavone aglycone, and has better activity function.

The soybean protein content is 30.8-57.9%, and the soybean protein can be mainly classified into albumin (accounting for about 10%) and globulin (accounting for about 90%). According to immunological analysis, globulin contains 40% of glycinin and 30% of beta-conglycinin, which are main antigen proteins in soybean and are easy to cause adverse reactions such as allergy and the like. However, the two antigen proteins have stronger heat stability, and the heat treatment at the temperature of 100 ℃ has little influence on the antigen activity. The microbial inoculum obtained by the scientific compatibility can also reduce the antigen activity of the soybean yoghourt, thereby improving the nutritional characteristics of the soybean product.

The invention also provides application of the microbial inoculum in preparation of the bean yoghourt.

When the microbial inoculum is used for preparing the soybean yoghourt, the soybean isoflavone glycoside can be converted into soybean isoflavone aglycone so as to play stronger biological activity and health care effect; meanwhile, the bean yoghourt prepared by the microbial inoculum has milk fragrance and bean fragrance, basically eliminates beany flavor, and has better flavor compared with the existing bean yoghourt products on the market.

Preferably, the method for preparing the bean yoghourt comprises the following steps: inoculating the microbial inoculum into the sterilized mixed soybean milk, and fermenting.

Preferably, the inoculation amount of the microbial inoculum in the mixed soybean milk is 3.0 multiplied by 10⁷～9.6×10¹⁰cfu/L。

Preferably, the temperature of the fermentation is 25-45 ℃. The fermentation time is 5-24 h.

Preferably, the fermentation is followed by a cold storage after-ripening treatment. More preferably, the refrigerating temperature is 3-5 ℃. The time for cold storage is preferably 6 hours or more than 6 hours.

Preferably, the mixed soybean milk is mainly prepared by mixing a milk matrix and a soybean matrix. More preferably, the mixed soybean milk further comprises a sweetener, and the sweetener is preferably sucrose.

Preferably, the milk base comprises any one or more of fresh milk, powdered milk and condensed milk.

Preferably, the soy base comprises any one or more of fresh soy milk and soy flour.

Preferably, the preparation method of the mixed soybean milk comprises the following steps: mixing 1-8 parts by weight of milk powder, 3-11 parts by weight of bean flour and 1-12 parts by weight of cane sugar with drinking water to obtain 100 parts by weight of materials, and uniformly stirring to completely dissolve the materials to obtain mixed soybean milk; or the preparation method of the mixed soybean milk comprises the following steps: mixing 30-50 parts by weight of fresh milk, 40-70 parts by weight of fresh soybean milk and 1-12 parts by weight of sucrose to obtain 100 parts by weight of materials, and uniformly stirring to completely dissolve the materials to obtain mixed soybean milk; or the preparation method of the mixed soybean milk comprises the following steps: mixing 1-8 parts by weight of milk powder, 40-60 parts by weight of fresh soybean milk and 1-12 parts by weight of cane sugar with drinking water to obtain 100 parts by weight of material, and uniformly stirring to completely dissolve the material to obtain mixed soybean milk; or the preparation method of the mixed soybean milk comprises the following steps: 30-50 parts by weight of fresh milk, 3-11 parts by weight of bean flour and 1-12 parts by weight of cane sugar are mixed with drinking water to obtain 100 parts by weight of materials, and the materials are uniformly stirred to be completely dissolved to obtain the mixed soybean milk.

The fresh soybean milk is preferably prepared according to a soybean-water weight ratio of 1: 6-1: 10.

The invention mixes the milk matrix and the bean matrix, and the nutritional components of the product are more comprehensive and balanced.

Compared with the prior art, the invention has the beneficial effects that:

the invention separates and identifies the strains with the suitability of the soy milk, obtains the microbial inoculum through reasonable compatibility, ferments the mixed soy milk, can obtain the soy milk with good curd effect and good flavor, can remove part of antigen protein, biologically converts the soy isoflavone, has high content of free soy isoflavone aglycone, and improves the biological activity and the biological utilization rate of the soy isoflavone in the organism.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the comparison of different strains in Experimental example 2 of the present invention with aglycone-producing soybean isoflavones;

FIG. 2 is a graph showing the comparison of the content of the antigen protein after the fermentation of soybean milk by different strains in Experimental example 2 of the present invention;

FIG. 3 is a graph showing the content of soy isoflavones in the mixed soymilk before fermentation in example 3 of the present invention; wherein D represents daidzin, G represents genistin, DE represents daidzein, and GE represents genistein;

FIG. 4 is a graph showing the content of soy isoflavones in soy yogurt obtained by fermentation using a microbial inoculum in example 3 of the present invention;

FIG. 5 is a graph showing the change in the isoflavone content before and after fermentation with the microbial inoculum in example 3 of the present invention.

The bean yogurt adaptive strain provided by the application comprises the following components:

a) lactococcus lactis (Lactococcus lactis), strain number is SCB0469, and the deposit number is: CGMCC 17619;

b) lactobacillus rhamnosus (Lactobacillus rhamnosus), strain number SCB0119, accession number: CGMCC 17618;

c) lactobacillus helveticus (Lactobacillus helveticus), strain number SCB0641, collection number: CGMCC 17620;

are all preserved in China general microbiological culture Collection center, and the preservation addresses are as follows: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the strains were detected as viable by the depository at 24/4.2019 and deposited.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The lactobacillus rhamnosus SCB0119 is separated from Sichuan pickle;

lactococcus lactis SCB0469 of the present invention was isolated from Russian kefir;

the lactobacillus helveticus SCB0641 of the present invention is isolated from tibetan pleurotus nebrodensis.

The streptococcus thermophilus, the lactobacillus plantarum, the lactobacillus casei and the lactobacillus paracasei adopted in the specific embodiment of the invention can be: streptococcus thermophilus CGMCC1.6472, Lactobacillus plantarum CGMCC1.573, Lactobacillus casei CGMCC1.8727, and Lactobacillus paracasei CGMCC 1.9088.

Example 1

The embodiment provides a soybean yoghourt adaptive microbial inoculum, which comprises four strains of lactococcus lactis SCB0469, Lactobacillus rhamnosus SCB0119, Streptococcus thermophilus and Lactobacillus helveticus SCB0641, wherein the ratio of the lactococcus lactis SCB0469, the Lactobacillus rhamnosus SCB0119, the Streptococcus thermophilus and the Lactobacillus helveticus SCB0641 is 2.0 multiplied by 10 in terms of the number of active bacteria⁹﹕4.0×10⁹﹕2.5×10⁸﹕8.0×10⁹。

The four strains are mixed in proportion to obtain the bean yoghourt adaptive microbial inoculum.

The method for preparing the bean yoghourt by using the microbial inoculum comprises the following steps:

(1) taking 2kg of milk powder, 9kg of pure soybean powder and 6kg of cane sugar, adding 100kg of drinking water, and fully and uniformly stirring to completely dissolve the materials to obtain mixed soybean milk; performing pasteurization on the mixed soybean milk at 90 deg.C for 20 min;

(2) cooling the sterilized mixed soybean milkCooling to 30-40 ℃, and inoculating a microbial inoculum; specifically, the bean yoghourt adaptive microbial inoculum is inoculated into the cooled mixed bean milk, and the total inoculation amount of the microbial inoculum is 8.5 multiplied by 10⁹cfu/L；

(3) Standing the inoculated mixed soybean milk, and fermenting at constant temperature, wherein the fermentation temperature is 42 ℃, and the fermentation time is 8 hours; and after fermentation is finished, refrigerating and ripening, wherein the refrigerating temperature is 3-5 ℃, and the refrigerating time is more than 12 hours, so that the bean yoghourt is obtained.

Example 2

The embodiment provides a soybean yoghourt adaptive microbial inoculum, which comprises three strains of lactococcus lactis SCB0469, streptococcus thermophilus and lactobacillus helveticus SCB0641, wherein the ratio of the lactococcus lactis SCB0469, the streptococcus thermophilus and the lactobacillus helveticus SCB0641 is 1.2 multiplied by 10 in terms of the number of live bacteria⁷﹕3.0×10⁶﹕1.8×10⁷。

The three strains are mixed in proportion to obtain the soy yogurt adaptive microbial inoculum.

(1) taking 3kg of milk powder, 8kg of pure soybean powder and 6kg of cane sugar, adding 100kg of drinking water, and fully and uniformly stirring to completely dissolve the materials to obtain mixed soybean milk; performing pasteurization on the mixed soybean milk at 90 deg.C for 20 min;

(2) cooling the sterilized mixed soybean milk to 30-40 ℃, and inoculating a microbial inoculum; specifically, the bean yoghourt adaptive microbial inoculum is inoculated into the cooled mixed bean milk, and the total inoculation amount of the microbial inoculum is 3 multiplied by 10⁷cfu/L；

(3) Standing the inoculated mixed soybean milk, and fermenting at constant temperature for 24h at 25 ℃; and after fermentation is finished, refrigerating and ripening, wherein the refrigerating temperature is 3-5 ℃, and the refrigerating time is more than 12 hours, so that the bean yoghourt is obtained.

Example 3

The embodiment provides a soybean yoghourt adaptive microbial inoculum, which comprises three strains of lactobacillus rhamnosus SCB0119, lactococcus lactis SCB0469 and streptococcus thermophilus, and the lactobacillus rhamnosus SCB0119 and the lactococcus lactis SCB0469 are counted by the number of the viable bacteriaThe ratio of Streptococcus thermophilus to Streptococcus thermophilus is 6.0X 10⁹﹕3.5×10⁹﹕2.0×10⁸。

(1) adding 42kg of drinking water into 7kg of milk powder, 58kg of fresh soybean milk and 2kg of cane sugar, and fully and uniformly stirring to completely dissolve the materials to obtain mixed soybean milk; performing pasteurization on the mixed soybean milk at 90 deg.C for 20 min;

(2) cooling the sterilized mixed soybean milk to 30-40 ℃, and inoculating a microbial inoculum; specifically, the bean yoghourt adaptive microbial inoculum is inoculated into the cooled mixed bean milk, and the total inoculation amount of the microbial inoculum is 9.5 multiplied by 10⁹cfu/L；

(3) Standing the inoculated mixed soybean milk, and fermenting at constant temperature for 12h at the fermentation temperature of 30 ℃; and after fermentation is finished, refrigerating and after-ripening, wherein the refrigerating temperature is 3-5 ℃, and the refrigerating and after-ripening time is more than 12 hours, so as to obtain the bean yoghourt.

Example 4

The embodiment provides a soybean yoghourt adaptive microbial inoculum, which comprises five strains of lactococcus lactis SCB0469, Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, Streptococcus thermophilus and Lactobacillus paracasei, wherein the ratio of the lactococcus lactis SCB0469, the Lactobacillus rhamnosus SCB0119, the Lactobacillus helveticus SCB0641, the Streptococcus thermophilus and the Lactobacillus paracasei is 1.5 multiplied by 10 in terms of the number of active bacteria⁸﹕6.5×10⁷﹕4.3×10⁸﹕2.0×10⁷﹕7.0×10⁹。

The five strains are mixed in proportion to obtain the bean yoghourt adaptive microbial inoculum.

(1) mixing 32kg of fresh milk, 68kg of fresh soybean milk and 10kg of sucrose, and fully and uniformly stirring to completely dissolve the materials to obtain mixed soybean milk; performing pasteurization on the mixed soybean milk at 90 deg.C for 20 min;

(2) cooling the sterilized mixed soybean milk to 30-40 ℃, and inoculating a microbial inoculum; specifically, the bean yoghourt adaptive microbial inoculum is inoculated into the cooled mixed bean milk, and the total inoculation amount of the microbial inoculum is 7.6 multiplied by 10⁹cfu/L；

(3) Standing the inoculated mixed soybean milk, and fermenting at constant temperature, wherein the fermentation temperature is 35 ℃, and the fermentation time is 13.5 hours; and after fermentation is finished, refrigerating and ripening, wherein the refrigerating temperature is 3-5 ℃, and the refrigerating time is more than 12 hours, so that the bean yoghourt is obtained.

Example 5

The embodiment provides a soybean yoghourt adaptive microbial inoculum, which comprises three strains of lactobacillus casei, lactococcus lactis SCB0469 and streptococcus thermophilus, wherein the ratio of the lactobacillus casei, the lactococcus lactis SCB0469 to the streptococcus thermophilus is 4.5 multiplied by 10 in terms of the number of live bacteria⁹﹕1.5×10⁹﹕4.0×10⁷。

This example refers to the method of making soy yogurt of example 2, with the only difference that: the inoculum size using this example was 6X 10⁹cfu/L, and the fermentation time is 18h, so that the bean yoghourt is prepared.

Example 6

The embodiment provides a soybean yoghourt adaptive microbial inoculum, which comprises three strains of lactobacillus paracasei, lactococcus lactis SCB0469 and streptococcus thermophilus, wherein the ratio of the lactobacillus paracasei, the lactococcus lactis SCB0469 to the streptococcus thermophilus is 4.5 multiplied by 10 in terms of the number of live bacteria⁹﹕3.5×10⁸﹕4.0×10⁸。

This example refers to the method of making soy yogurt of example 3, except that the inoculum size of this example was 5.3X 10⁹cfu/L, and preparing the bean yoghourt.

Example 7

The embodiment provides a bean yogurt adaptive microbial inoculum which comprises three strains of lactobacillus plantarum, lactococcus lactis SCB0469 and streptococcus thermophilusThe ratio of Lactobacillus plantarum to lactococcus lactis SCB0469 to Streptococcus thermophilus was 4.5X 10 in terms of viable count⁸﹕5.5×10⁹﹕4.0×10⁸。

This example refers to the method of making soy yogurt of example 3, except that the inoculum size of this example was 5.6X 10⁹cfu/L, fermenting for 18h, and preparing the bean yoghourt.

Example 8

The embodiment provides a bean yogurt adaptive microbial inoculum, which comprises four strains of lactobacillus helveticus SCB0641, lactobacillus paracasei, lactococcus lactis SCB0469 and streptococcus thermophilus, wherein the ratio of the lactobacillus helveticus SCB0641, the lactobacillus paracasei, the lactococcus lactis SCB0469 and the streptococcus thermophilus is 5.0 multiplied by 10 in terms of the number of living bacteria⁹﹕4.5×10⁸﹕8.5×10⁸﹕1.0×10⁹。

This example refers to the method of making soy yogurt of example 2, except that the inoculum size of this example was 7.3X 10⁹cfu/L, fermenting for 18h, and preparing the bean yoghourt.

Example 9

The embodiment provides a soybean yoghourt adaptive microbial inoculum, which comprises seven strains of lactobacillus rhamnosus SCB0119, lactobacillus helveticus SCB0641, lactobacillus paracasei, lactobacillus lactis SCB0469, lactobacillus plantarum, lactobacillus casei and streptococcus thermophilus, wherein the ratio of the lactobacillus rhamnosus SCB0119, the lactobacillus helveticus SCB0641, the lactobacillus paracasei, the lactobacillus lactis SCB0469, the lactobacillus plantarum, the lactobacillus casei and the streptococcus thermophilus is 3.5 multiplied by 10 in terms of the number of the viable bacteria⁸﹕4.0×10⁹﹕4.5×10⁸﹕1.5×10⁸﹕3.0×10⁸﹕5.5×10⁹﹕1.0×10⁸。

Mixing the seven strains in proportion to obtain the bean yoghourt adaptive microbial inoculum.

This example refers to example 2Method for preparing a soy yogurt, the only difference being that the inoculum size used in this example was 1.13X 10¹⁰cfu/L, fermenting for 16h, and preparing the bean yoghourt.

Example 10

The embodiment provides a soybean yoghourt adaptive microbial inoculum, which comprises seven strains of lactobacillus rhamnosus SCB0119, lactobacillus helveticus SCB0641, lactobacillus paracasei, lactobacillus lactis SCB0469, lactobacillus plantarum, lactobacillus casei and streptococcus thermophilus, wherein the ratio of the lactobacillus rhamnosus SCB0119, the lactobacillus helveticus SCB0641, the lactobacillus paracasei SCB0469, the lactobacillus plantarum, the lactobacillus casei and the streptococcus thermophilus is 2.5 multiplied by 10 in terms of the number of live bacteria⁹﹕4.0×10⁸﹕5.5×10⁹﹕2.5×10⁹﹕5.0×10⁹﹕5.5×10⁸﹕3.0×10⁹。

This example refers to the method of making soy yogurt of example 2, except that the total inoculum size using this example is 1.9X 10¹⁰cfu/L, fermenting for 16h, and preparing the bean yoghourt.

Comparative example 1

Comparative example 1 the preparation process of example 3 was referenced, except that the inoculated fermentation broth was CGMCC1.16075 and Streptococcus thermophilus CGMCC1.6472 at a ratio of 5.5X 10⁹﹕4.0×10⁹Inoculation amount of 9.5X 10⁹cfu/L。

Experimental example 1

The experimental method comprises the following steps:

the lactococcus lactis (all isolated from kefir) stored in the laboratory was removed from the ultra-low temperature refrigerator and inoculated into 8mL of MRS liquid medium by using a sterile pipette in an inoculum size of 1% (v/v) and cultured at 37 ℃ for 12 hours, and passaging was performed once in the same manner.

Then, the activated liquid strain is inoculated into the mixed soymilk (5% pure soybean powder, 5% whole milk powder, 8% white granulated sugar, pasteurization at 90 ℃ for 20min, cooling to 40 ℃) according to the inoculation amount of 1% (v/v), and placed at 25 ℃ for culturing for 24 h.

And observing and recording the fermentation condition, measuring the pH value of the fermented soybean yoghourt by using a pH meter, observing the curd condition by naked eyes, judging the viscosity by drawing the toothpick, and evaluating the smell of the soybean yoghourt in a sensory manner.

The experimental results are as follows:

experimental example 1 the fermented effect of the soybean yogurt with lactococcus lactis was screened, and the results are shown in Table 1 below.

TABLE 1 screening of fermented Soybean yogurt Strain lactococcus lactis

Note: "+" indicates curd, "-" indicates no curd; a sticky condition "+" indicates sticky and "-" indicates not sticky; the savoury soymilk taste is a mixed taste of the flavour of soymilk and the flavour of sour milk in yoghurt.

As can be seen from Table 1, the screened lactococcus lactis fermented soy yogurt at 25 ℃ for 24h, the curd was poor, and only SCB0427 and SCB0469 fermented soy yogurt curds. The strain with stronger acid production is SCB0427, and the pH value is 5.25. The fermented soybean yoghourt has poor viscosity, most of the fermented soybean yoghourt has soybean milk flavor, and the part of the fermented soybean yoghourt has sour and fragrant soybean milk flavor, wherein the fermented soybean milk has the sour and fragrant soybean milk flavor of SCB0427, SCB0431, SCB0433, SCB0449, SCB0469 and SCB 0477. By combining the indexes, the selected SCB0469 fermented soybean yoghourt has better curd effect, the pH values are respectively 5.25 and 5.46, the soybean yoghourt has proper odor, and the soybean yoghourt is suitable for being fermented.

Experimental example 2

Experimental example 2 conditions of aglycone-producing soybean isoflavones and contents of antigen proteins after fermented soybean milk were tested according to different strains, as shown in FIGS. 1 and 2.

The test method comprises the following steps:

soaking soybeans: adding 0.5% NaHCO to the washed soybeans₃Soaking the solution at room temperature for 10 hr at a ratio of 1: 3 to make the soybean fully swell.

Blanching: washing the soaked soybeans with distilled water, and blanching at 85-90 deg.C for 10 min.

Grinding: grinding the scalded soybean with beating machine at ratio of 1: 6 for 2min, filtering with 4 layers of gauze, and removing bean dregs.

Sterilization and inoculation fermentation: heating soybean milk to 100 deg.C, boiling for 6min, keeping the temperature at 90 deg.C for 15-20min, cooling to 30 deg.C, inoculating various lactobacillus strains (culture solution after three generations of MRS liquid culture medium activation) according to 2%, shaking, mixing, standing at 25 deg.C, fermenting for 24h, and measuring the contents of two antigen proteins with the fermentation liquid as follows.

The method for measuring the soybean antigenic protein comprises the following steps:

extraction of soybean antigen protein sample: taking 1.0mL of a sample to be detected, soaking the sample in 20.0mL of 0.03mol/L Tris-HCl (pH 8.0 and including 0.01mol/L beta-mercaptoethanol) buffer solution, soaking the sample on a shaking table at the temperature of 28 ℃ for 1h at the speed of 150r/min, then centrifuging the sample at the speed of 4000r/min for 20min, and taking a supernatant for later use.

And (3) detecting the immunological activity: the assay of glycinin was performed according to the method in the purchased antigenic protein ELISA kit (96 wells). Soybean antigenic protein ELISA kit (purchased from Yu duo Biotech, Inc. of Shanghai).

As can be seen from FIG. 1, SCB0641 can significantly increase the content of Daidzein (DE) and Genistin (GE) in fermented soy isoflavone and increase the content of free soy isoflavone aglycone, thereby increasing the bioactivity and bioavailability of soy isoflavone in vivo, compared with other strains.

As can be seen from FIG. 2, SCB0119 can obviously reduce the antigenic contents of two antigenic proteins, namely beta-conglycinin (SPAg) and glycinin (SPA), and the strain can also improve the content of genistin.

Experimental example 3

In order to illustrate the fermentation effect of the microbial inoculum of the invention on the mixed soybean milk, taking example 3 as an example, the content of the soybean isoflavone in the mixed soybean milk before fermentation and the soybean yogurt obtained after fermentation is measured, and the test results are shown in fig. 3-5, fig. 3 is a graph of the content of the soybean isoflavone in the mixed soybean milk before fermentation, fig. 4 is a graph of the content of the soybean isoflavone in the soybean yogurt obtained after fermentation, and fig. 5 is a graph of the change of the content of the soybean isoflavone before and after fermentation. It can be known from the figure that before fermentation, the content of daidzin (D) and genistin (G) is higher, after fermentation, the content of daidzin and genistin is relatively reduced, and meanwhile, the content of Daidzein (DE) and Genistein (GE) is obviously improved, which shows that the microbial inoculum of the invention ferments the mixed soybean milk, can biologically convert the soybean isoflavone, improves the content of free soybean isoflavone aglycone, and further improves the biological activity and bioavailability of the soybean isoflavone in the body.

Experimental example 4

In order to comparatively illustrate the fermentation effect of different microbial agents of the present invention on mixed soybean milk, the content of soybean isoflavone and the degradation of antigen protein in the soybean yogurt obtained by fermentation in examples 1 to 10 and comparative example 1 were tested, and the test results are shown in table 2.

TABLE 2 content of soy isoflavones in different soy yoghurts (unit: μ g/mL soy yoghurt)

Numbering	Daidzin	Genistin	Daidzein	Genistein	D value
						Example 1	78.3±2.7	75.9±1.2	-	0.43±0.04	1.53
Example 2	81.1±4.2	78±0.3	-	0.11±0.02	1.32
						Example 3	52.2±2.3	45.9±2.2	12.2±1.2	15.8±2.8	14.52
Example 4	65.3±2.3	69.6±5.4	9.3±0.8	6.9±0.2	1.22
						Example 5	82.2±3.4	75.3±4.8	-	0.6±0.04	0.13
Example 6	76.3±4.5	76.8±0.4	0.4±0.04	-	-0.05
						Example 7	77.3±0.9	75.5±6.2	3.0±0.43	0.4±0.02	0.22
Example 8	57.2±2.9	51.3±0.8	8.3±0.12	10.2±0.44	-0.43
						Example 9	60.4±2.2	49.3±4.3	10.2±0.5	14.3±3.2	5.66
Example 10	43.4±3.4	46.2±4.5	16.0±3.2	20.2±4.8	12.26
						Comparative example 1	82.3±2.1	76.3±5.4	-	-	0.11

Note: calculation of D value: (content of antigen protein in soymilk before fermentation-content of antigen protein in soymilk after fermentation)/content of antigen protein in soymilk before fermentation X100

As can be seen from Table 2, the soy yogurt adaptive strain of the present invention can biologically convert soy isoflavones, significantly increase the content of free soy isoflavone aglycones, and increase the biological activity and bioavailability of soy isoflavones in the body.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> southern China university of agriculture

<120> soy yogurt adaptive strain, microbial inoculum and application thereof

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1380

<212> DNA

<213> Lactobacillus rhamnosus

<400> 1

gcttgcatct tgatttaatt ttgaacgagt ggcggacggg tgagtaacac gtgggtaacc 60

tgcccttaag tgggggataa catttggaaa cagatgctaa taccgcataa atccaagaac 120

cgcatggttc ttggctgaaa gatggcgtaa gctatcgctt ttggatggac ccgcggcgta 180

ttagctagtt ggtgaggtaa cggctcacca aggcaatgat acgtagccga actgagaggt 240

tgatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 300

atcttccaca atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag aaggctttcg 360

ggtcgtaaaa ctctgttgtt ggagaagaat ggtcggcaga gtaactgttg tcggcgtgac 420

ggtatccaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 480

gcaagcgtta tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 540

gtgaaagccc tcggcttaac cgaggaagtg catcggaaac tgggaaactt gagtgcagaa 600

gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 660

ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagcatg ggtagcgaac 720

aggattagat accctggtag tccatgccgt aaacgatgaa tgctaggtgt tggagggttt 780

ccgcccttca gtgccgcagc taacgcatta agcattccgc ctggggagta cgaccgcaag 840

gttgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 900

gaagcaacgc gaagaacctt accaggtctt gacatctttt gatcacctga gagatcaggt 960

ttccccttcg ggggcaaaat gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1020

tgttgggtta agtcccgcaa cgagcgcaac ccttatgact agttgccagc atttagttgg 1080

gcactctagt aagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1140

atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaga 1200

ccgcgaggtc aagctaatct cttaaagcca ttctcagttc ggactgtagg ctgcaactcg 1260

cctacacgaa gtcggaatcg ctagtaatcg cggatcagca cgccgcggtg aatacgttcc 1320

cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccga agccggtggc 1380

<210> 2

<211> 1419

<212> DNA

<213> Lactococcus lactis

<400> 2

gagcgccctc cttgcggtta ggcaacctac ttcgggtact cccaactccc gtggtgtgac 60

gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc gcgattacta 120

gcgattccga cttcatgtag gcgagttgca gcctacaatc cgaactgaga atggttttaa 180

gagattagct aaacatcact gtctcgcgac tcgttgtacc atccattgta gcacgtgtgt 240

agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttatca 300

ccggcagtct cgttagagtg cccaacttaa tgatggcaac taacaatagg ggttgcgctc 360

gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420

atcccgtgtc ccgaaggaac ttcctatctc taggaatagc acgagtatgt caagacctgg 480

taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540

tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggagtgc ttattgcgtt 600

agctgcgata cagagaactt atagctccct acatctagca ctcatcgttt acggcgtgga 660

ctaccagggt atctaatcct gtttgctccc cacgctttcg agcctcagtg tcagttacag 720

gccagagagc cgctttcgcc accggtgttc ctccatatat ctacgcattt caccgctaca 780

catggaattc cactctcctc tcctgcactc aagtctacca gtttccaatg catacaatgg 840

ttgagccact gccttttaca ccagacttaa taaaccacct gcgctcgctt tacgcccaat 900

aaatccggac aacgctcggg acctacgtat taccgcggct gctggcacgt agttagccgt 960

ccctttctgg gtagttaccg tcacttgatg agctttccac tctcaccaac gttcttctct 1020

accaacagag ttttacgatc cgaaaacctt cttcactcac gcggcgttgc tcggtcagac 1080

tttcgtccat tgccgaagat tccctactgc tgcctcccgt aggagtttgg gccgtgtctc 1140

agtcccaatg tggccgatca ccctctcagg tcggctatgt atcatcgcct tggtgagcct 1200

ttacctcacc aactagctaa tacaacgcgg gatcatcttt gagtgatgca attgcatctt 1260

tcaaacttaa aacttgtgtt taaagttttt atgcggtatt agcattcgtt tccaaatgtt 1320

gtcccccgct caaaggcaga ttccccacgc gttactcacc cgttcgctgc tcatccagtt 1380

ggtacaagta ccaaccttca gcgctcaact tgcatgtat 1419

<210> 3

<211> 1409

<212> DNA

<213> Lactobacillus helveticus

<400> 3

ggccaccggc tttgggcatt gcagacttcc atggtgtgac gggcggtgtg tacaaggccc 60

gggaacgtat tcaccgcggc gttctgatcc gcgattacta gcgattccag cttcgtgcag 120

tcgagttgca gactgcagtc cgaactgaga acagctttca gagattcgct tgccttcgca 180

ggctcgcttc tcgttgtact gcccattgta gcacgtgtgt agcccaggtc ataaggggca 240

tgatgacttg acgtcatccc caccttcctc cggtttgtca ccggcagtct cattagagtg 300

cccaacttaa tgctggcaac taataataag ggttgcgctc gttgcgggac ttaacccaac 360

atctcacgac acgagctgac gacagccatg caccacctgt cttagcgtcc ccgaagggaa 420

ctcctaatct cttaggatgg cactagatgt caagacctgg taaggttctt cgcgttgctt 480

cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcctt tgagtttcaa 540

ccttgcggtc gtactcccca ggcggagtgc ttaatgcgtt agctgcagca ctgagaggcg 600

gaaacctccc aacacttagc actcatcgtt tacggcatgg actaccaggg tatctaatcc 660

tgttcgctac ccatgctttc gagcctcagc gtcagttgca gaccagagag ccgccttcgc 720

cactggtgtt cttccatata tctacgcatt ccaccgctac acatggagtt ccactctcct 780

cttctgcact caagaaaaac agtttccgat gcaattcctc ggttaagccg agggctttca 840

catcagactt attcttccgc ctgcgctcgc tttacgccca ataaatccgg acaacgcttg 900

ccacctacgt attaccgcgg ctgctggcac gtagttagcc gtgactttct ggttgattac 960

cgtcaaataa aggccagtta ctacctctat ccttcttcac caacaacaga gctttacgat 1020

ccgaaaacct tcttcactca cgcggcgttg ctccatcaga cttgcgtcca ttgtggaaga 1080

ttccctactg ctgcctcccg taggagtttg ggccgtgtct cagtcccaat gtggccgttc 1140

agtctctcaa ctcggctatg catcattgcc ttggtaagcc gttaccttac caactagcta 1200

atgcaccgcg gggccatccc atagcgacag cttacgccgc cttttataag ctgatcatgc 1260

gatctgcttt attatccggt attagcacct gtttccaagt ggtatcctag actatggggc 1320

aggttcccca cgtgttactc acccatccgc cgctcgcgtc cccagcgtca ttaccgaagt 1380

aaatctgctg gttctgctcg ctcgacttg 1409

Claims

1. soy yogurt suitable bacterial strain, is characterized in that, described bacterial strain is:

a) Lactococcus lactis SCB0469, deposited in the China Microbial Culture Collection Center, the preservation number is: CGMCC17619; the preservation time is: April 24, 2019.

2. The inoculum comprising the soy yogurt-suitable strain of claim 1.

3. microbial inoculum according to claim 2, is characterized in that, also comprises any one or both in following bacterial strain in described microbial inoculum;

b) Lactobacillus rhamnosus SCB0119, deposited in the China Microbial Culture Collection Center, preservation number: CGMCC17618; preservation time: April 24, 2019;

c) Lactobacillus helveticus SCB0641, deposited in the China Microbial Culture Collection Center, with the preservation number: CGMCC17620; preservation time: April 24, 2019.

4. The bacterial agent according to claim 2 or 3, further comprising any one or more of Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus paracasei.

5. microbial inoculum according to claim 4 is characterized in that, in the described microbial inoculum, in terms of viable count, the ratio of Lactobacillus rhamnosus SCB0119, Lactococcus lactis SCB0469 and Lactobacillus helveticus SCB0641 is (0～ 1.22×10 ¹⁰ ): (1.0×10 ⁶ to 3.60×10 ¹⁰ ): (0 to 8.55×10 ¹⁰ ).

6. microbial inoculum according to claim 4, is characterized in that, in described microbial inoculum, counted by viable count, lactobacillus rhamnosus SCB0119, lactococcus lactis SCB0469, lactobacillus helveticus SCB0641, streptococcus thermophilus The ratio is (0～1.22×10 ¹⁰ ):(1.0×10 ⁶ ～3.60×10 ¹⁰ ):(0～8.55×10 ¹⁰ ):(1.0×10 ⁶ ～4.54×10 ⁹ ).

7 . The bacterial agent according to claim 3 , wherein the bacterial agent comprises Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, Lactococcus lactis SCB0469 and Lactobacillus paracasei. 8 .

8. microbial inoculum according to claim 7, is characterized in that, in described microbial inoculum, counted by viable count, Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, Lactococcus lactis SCB0469, Lactobacillus paracasei The ratio is (6.5×10 ⁷ ～3.0×10 ⁹ ): (1.5×10 ⁸ ～5.0×10 ⁹ ): (1.5×10 ⁸ ～3.0×10 ⁹ ): (4.0×10 ⁸ ～7.5×10 ⁹ ).

The microbial inoculum according to claim 7, characterized in that, the microbial inoculum further comprises Streptococcus thermophilus.

10. microbial inoculum according to claim 9, is characterized in that, in described microbial inoculum, by viable count, Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, Lactococcus lactis SCB0469, Lactobacillus paracasei, The ratio of Streptococcus thermophilus was (6.5×10 ⁷ ～2.5×10 ⁹ ): (4.0×10 ⁸ ～4.0×10 ⁹ ):(1.5×10 ⁸ ～2.5×10 ⁹ ):(4.5×10 ⁸ ～7.0 ×10 ⁹ ): (2.0×10 ⁷ to 3.0×10 ⁹ ).

11. The inoculum according to claim 9, wherein the inoculum also comprises Lactobacillus plantarum and/or Lactobacillus casei.

12. microbial inoculum according to claim 11, is characterized in that, in described microbial inoculum, by viable count, Lactobacillus rhamnosus SCB0119, Lactobacillus helveticus SCB0641, Lactococcus lactis SCB0469, Lactobacillus paracasei, The ratio of Streptococcus thermophilus, Lactobacillus plantarum and Lactobacillus casei is (6.5×10 ⁷ ～2.5×10 ⁹ ): (4.0×10 ⁸ ～4.0×10 ⁹ ): (1.5×10 ⁸ ～2.5×10 ⁹ ) : (4.5×10 ⁸ to 7.0×10 ⁹ ): (2.0×10 ⁷ to 3.0×10 ⁹ ): (3.0×10 ⁸ to 5.0×10 ⁹ ): (5.5×10 ⁸ to 5.5×10 ⁹ ).

The microbial inoculum according to claim 3, wherein the microbial inoculum comprises Lactococcus lactis SCB0469 and Lactobacillus helveticus SCB0641.

The microbial inoculum according to claim 13, characterized in that, in the microbial inoculum, the ratio of Lactococcus lactis SCB0469 to Lactobacillus helveticus SCB0641 is (1×10 ⁷ to 2.5×10 ⁹ in terms of viable cell counts) ): (1.5×10 ⁷ to 4.0×10 ⁹ ).

The microbial inoculum according to claim 13, wherein the microbial inoculum further comprises Streptococcus thermophilus.

16. microbial inoculum according to claim 15, is characterized in that, in described microbial inoculum, by viable count, the ratio of Lactococcus lactis SCB0469, Lactobacillus helveticus SCB0641, Streptococcus thermophilus is (1 × 10 ⁷ ～2.5×10 ⁹ ): (1.5×10 ⁷ ～4.0×10 ⁹ ): (3×10 ⁶ ～3×10 ⁹ ).

17. The bacterial preparation according to claim 13, wherein the bacterial preparation further comprises Lactobacillus paracasei.

18. microbial inoculum according to claim 17, is characterized in that, in described microbial inoculum, by viable count, the ratio of Lactobacillus helveticus SCB0641, Lactobacillus paracasei, Lactococcus lactis SCB0469 is (3.0×10 ⁸ ～5.0×10 ⁹ ): (4.0×10 ⁸ ～5.5×10 ⁹ ): (1.5×10 ⁸ ～2.5×10 ⁹ ).

The microbial inoculum according to claim 3, wherein the microbial inoculum comprises Lactobacillus rhamnosus SCB0119 and Lactococcus lactis SCB0469.

20 . The bacterial agent according to claim 19 , wherein, in the bacterial agent, the ratio of Lactobacillus rhamnosus SCB0119 to Lactococcus lactis SCB0469 is (6.5×10 ⁷ to 6× 10 ⁹ ): (1.5×10 ⁸ to 3.5×10 ⁹ ).

21. The bacterial agent according to claim 19, wherein the bacterial agent further comprises Streptococcus thermophilus.

22. The microbial inoculum according to claim 21, wherein in the microbial inoculum, the ratio of Lactobacillus rhamnosus SCB0119, Lactococcus lactis SCB0469, and Streptococcus thermophilus is (6.5× 10 ⁷ to 6×10 ⁹ ): (1.5×10 ⁸ to 3.5×10 ⁹ ): (2×10 ⁷ to 3×10 ⁹ ).

23. The bacterial agent according to claim 2, wherein the bacterial agent comprises Lactobacillus casei and Lactococcus lactis SCB0469.

24. The bacterial agent according to claim 23, wherein, in the bacterial agent, the ratio of Lactobacillus casei and Lactococcus lactis SCB0469 is (4×10 ⁸ to 8×10 ⁹ ) in terms of viable count. : (1×10 ⁸ to 2.5×10 ⁹ ).

25. The bacterial agent according to claim 23, wherein the bacterial agent further comprises Streptococcus thermophilus.

The microbial inoculum according to claim 25, characterized in that, in the microbial inoculum, the ratio of Lactobacillus casei, Lactococcus lactis SCB0469 and Streptococcus thermophilus is (5.5×10 ⁸ ~ 5.5 in terms of viable counts) ×10 ⁹ ): (1.5×10 ⁸ to 2.5×10 ⁹ ): (4.0×10 ⁷ to 3.0×10 ⁹ ).

27. The bacterial agent according to claim 2, wherein the bacterial agent comprises Lactobacillus paracasei and Lactococcus lactis SCB0469.

28. The bacterial preparation according to claim 27, wherein in the bacterial preparation, the ratio of Lactobacillus paracasei and Lactococcus lactis SCB0469 is (3.0 × 10 ⁸ to 7.0 × 10 ⁹ in terms of viable counts) ): (1.5×10 ⁸ to 2.5×10 ⁹ ).

29. The bacterial agent according to claim 27, wherein the bacterial agent further comprises Streptococcus thermophilus.

30. The bacterial agent according to claim 29, wherein, in the bacterial agent, the ratio of Lactobacillus paracasei, Lactococcus lactis SCB0469 and Streptococcus thermophilus is (4.5×10 ⁸ ~ 7.0×10 ⁹ ): (1.5×10 ⁸ to 2.5×10 ⁹ ): (2.0×10 ⁷ to 3.0×10 ⁹ ).

31. The inoculum according to claim 2, wherein the inoculum comprises Lactobacillus plantarum and Lactococcus lactis SCB0469.

32. The bacterial agent according to claim 31, wherein, in the bacterial agent, the ratio of Lactobacillus plantarum and Lactococcus lactis SCB0469 is (3.0×10 ⁸ to 8.0×10 ⁹ ) in terms of the number of viable cells. : (1.5×10 ⁸ to 8.0×10 ⁹ ).

33. The bacterial agent according to claim 31, wherein the bacterial agent further comprises Streptococcus thermophilus.

34. The bacterial agent according to claim 33, wherein, in the bacterial agent, the ratio of Lactobacillus plantarum, Lactococcus lactis SCB0469 and Streptococcus thermophilus is (3.0×10 ⁸ -5.0 ×10 ⁹ ): (1.5×10 ⁸ to 5.5×10 ⁹ ): (1.0×10 ⁸ to 3.0×10 ⁹ ).

35. The application of the bacterial agent according to any one of claims 2-34 in the production of soy yogurt.

36 . The application according to claim 35 , wherein the method for making the soy yogurt comprises: inoculating the microbial inoculum in the sterilized mixed soy milk to ferment. 37 .

37. The application according to claim 36, wherein the inoculum of the bacterial agent in the mixed soymilk is 3.0×10 ⁷ to 9.6×10 ¹⁰ cfu/L.

38. The application according to claim 36, wherein the fermentation temperature is 25-45°C; and the fermentation time is 5-24h.

39. The application according to claim 36, wherein the mixed soymilk is mainly made by mixing a milk base and a bean base.

40. The application according to claim 39, wherein the mixed soymilk further comprises a sweetener.

41. The use according to claim 39, wherein the milk base comprises any one or more of fresh milk, milk powder and condensed milk.

42. The application according to claim 39, wherein the bean base comprises any one or more of fresh soybean milk and soybean flour.