阅读说明：本技术 一种具有抗氧化功能的混合乳酸菌发酵乳其制备方法 (Mixed lactobacillus fermented milk with antioxidant function and preparation method thereof ) 是由 孙宁 谢秉锵 李仁芳 伍云 李婕 黄学正 石爱萍 李雯晓 黄丽 李玲 杨攀 于 2021-08-06 设计创作，主要内容包括：本发明涉及生物发酵领域,具体涉及一种具有抗氧化功能的混合乳酸菌发酵乳其制备方法。所述混合乳酸菌发酵乳采用益生乳酸菌L.fermentumPM22：P.acidilacticiPM44为1:2混合发酵制备而成。同时,本发明还提供了所述乳酸菌发酵乳的制备方法。本发明益生乳酸菌筛选自于传统发酵食品且为γ-溶血的安全菌株,同时具有耐胃肠条件、高抗氧化活性的特性。按一定配比益生混合乳酸菌与商业发酵剂复配使用,利用共生机制,不仅提高发酵乳的抗氧化特性,同时减少发酵时间和后酸化等不良影响。(The invention relates to the field of biological fermentation, and in particular relates to mixed lactobacillus fermented milk with an antioxidant function and a preparation method thereof. The mixed lactobacillus fermented milk adopts probiotic lactobacillus L.fermentumPM22: p. acidilacticipM44 is prepared by 1:2 mixed fermentation. Meanwhile, the invention also provides a preparation method of the lactobacillus fermented milk. The probiotic lactic acid bacteria sieve is selected from traditional fermented food and is a gamma-hemolytic safe bacterial strain, and has the characteristics of gastrointestinal condition resistance and high antioxidant activity. The probiotic mixed lactic acid bacteria and the commercial starter are compounded according to a certain proportion, and a symbiotic mechanism is utilized, so that the antioxidant property of the fermented milk is improved, and the adverse effects of fermentation time, post-acidification and the like are reduced.)

1. A natural probiotic starter, which comprises probiotic lactic acid bacteria L.fermentum PM22 and P.acidilactici PM44, wherein the preservation number of the L.fermentum PM22 is GDMCC No: 61678, the P.acidilactici PM44 has a deposit number of GDMCC No: 61705.

2. the natural probiotic starter culture of claim 1 wherein the volume ratio of the probiotic lactic acid bacteria is l.fermentum PM 22: acidilactici PM44 is 1:2 to 2: 1.

3. The natural probiotic starter culture of claim 1 wherein the volume ratio of the probiotic lactic acid bacteria is l.fermentum PM 22: acidilactici PM44 was 1: 2.

4. A mixed lactic acid bacteria fermented milk having a high antioxidant function, comprising the probiotic fermentation agent according to any one of claims 1 to 3.

5. The mixed lactic acid bacteria fermented milk according to claim 4, further comprising raw milk, commercial starter, sucrose.

6. The method for preparing the mixed lactic acid bacteria fermented milk with high antioxidant function according to claim 5, wherein the method comprises the following steps: 1) blending, namely adding 5-7% of cane sugar in percentage by mass into raw milk; 2) homogenizing, dissolving sucrose at 55-70 deg.C, homogenizing under 18-20 Mpa; 3) sterilizing at 90-95 deg.C for 8-11 min; 4) cooling; 5) inoculating, inoculating the probiotic starter culture of claim 2, while adding a commercial starter culture; 6) fermenting at 38-42 deg.C to acidity of 70-80T; 7) refrigerating; 8) and (5) after-ripening.

7. The method according to claim 6, wherein the raw milk is one or more of Holstein cow's milk, Juansan cow's milk, yak's milk, goat's milk, and buffalo's milk.

8. The preparation method according to claim 6, wherein the inoculation amount of the probiotic leavening agent is 3-5% by mass, and the inoculation amount of the commercial leavening agent is 0.003-0.008% by mass.

9. The method according to claim 6, wherein the commercial fermentation agent is a direct vat set fermentation agent comprising Lactobacillus bulgaricus and Streptococcus thermophilus.

Technical Field

The invention relates to the field of biological fermentation, and in particular relates to mixed lactobacillus fermented milk with an antioxidant function and a preparation method thereof.

Background

Fermented milk is generally classified into three types, i.e., normal, functional and flavor types, and flavor types are generally classified into a stirring type and a solidifying type. Compared with fresh cow milk, the fermented milk contains more abundant nutrient substances and sour, sweet and delicious taste, and is popular among people. The fermented milk also has unique effect on human body, such as extremely wide effect on health care of human body. For example: the fermented milk has rich food nutrition, and has effects of improving intestinal bacteria flora, promoting liver and kidney health, and preventing and treating other diseases. With the rapid development of production technology, people gradually deepen understanding about the functions of the fermented milk, such as nutrition and health, and meanwhile, the sales volume is continuously increased to present a rapid growth trend by continuously improving the flavor of the fermented milk. At present, the consumption of fermented milk by everyone in China is still low, so that the fermented milk product market in China has very good prospects. In recent years, fermented milk has been gradually developed to have a variety of tastes and tastes, but it still cannot satisfy consumers, and people have been pursuing health and nutrition. The emergence of functional foods is the mainstream trend, fermented milks with probiotic properties have successfully occupied the major market and the development of functional fermented dairy products has already been a development trend.

In recent years, the relationship between active oxygen and free radicals and diseases has been more and more emphasized, and the development of antioxidants corresponding to the active oxygen and free radicals has become an important subject of food science. From the viewpoint of food safety, natural antioxidant substances have incomparable advantages with synthetic antioxidants, and are now the targets pursued by food and medical experts today. Research shows that the lactobacillus has a plurality of physiological functions of regulating the intestines, inhibiting bacteria, resisting cancer, enhancing the immunity of the organism, resisting aging and the like. Therefore, lactic acid bacteria will have great market potential as antioxidants, both from the safety and nutritional and health value perspective. Lactic acid bacteria are generally present in vegetables, fruits, dairy products, meat products, aquatic products, in particular in various fermented foods. If the lactobacillus with the antioxidant activity can be screened from the food source angle and used for producing functional fermented food and health care products or developed into anti-aging medicines, the lactobacillus has positive effects on promoting the domestic probiotic industry and promoting the health of people, and can bring good economic and social benefits.

Disclosure of Invention

One of the objects of the present invention is: provides a mixed lactobacillus fermented milk with antioxidant function and a preparation method thereof:

in one aspect, the invention provides a natural probiotic starter, which comprises probiotic lactic acid bacteria L.fermentum PM22 and P.acidilactici PM44, and is stored in the Guangdong province collection center of microorganism strains in 26 months 5 and 2021, wherein the preservation number of the L.fermentum PM22 is GDMCC No: 61678, the P.acidilactici PM44 accession number is GDMCC No: 61705.

the volume ratio of the probiotic lactic acid bacteria is L.fermentum PM 22: acidilactici PM44 is 1:2 to 2: 1.

Preferably, the volume ratio of the probiotic lactic acid bacteria is L.fermentum PM 22: acidilactici PM44 was 1: 2.

In the period from 2018 to 2019, the laboratory performs lactic acid bacteria separation and screening work on the traditional fermented raw rice flour in Guangxi, and separates 318 strains of lactic acid bacteria cumulatively to identify 294 strains of lactic acid bacteria. In vitro functional probiotic properties of the isolated lactic acid bacteria were studied, and 2 lactic acid bacteria strains with good probiotic properties were obtained, as shown in table 1.

TABLE 12 lactic acid bacteria with good probiotic properties

In another aspect, the invention provides mixed lactic acid bacteria fermented milk with high antioxidant function, which comprises any one group of the probiotic leavening agents.

Further, the mixed lactic acid bacteria fermented milk with the high antioxidant function also comprises raw milk, a commercial starter and sucrose.

In another aspect, the invention further provides a method for preparing mixed lactobacillus fermented milk with high antioxidant function, wherein the method comprises the following steps: 1) blending, namely adding 5-7% of cane sugar in percentage by mass into raw milk; 2) homogenizing, dissolving sucrose at 55-70 deg.C, homogenizing under 18-20 Mpa; 3) sterilizing at 90-95 deg.C for 8-11 min; 4) cooling; 5) inoculating, inoculating the probiotic starter culture of claim 2, while adding a commercial starter culture; 6) fermenting at 38-42 deg.C to acidity of 70-80T; 7) refrigerating; 8) and (5) after-ripening.

The raw milk is one or more of Holstein cow milk, Juansan cow milk, yak milk, goat milk and buffalo milk.

Further, the inoculation amount of the probiotic leavening agent is 3-5% by mass, and the inoculation amount of the commercial leavening agent is 0.003-0.008% by mass.

Further, the commercial starter is a direct vat starter, especially one produced by Kehansen LimitedPreferably, the MiId 1.050U direct vat set starter contains Lactobacillus bulgaricus and Streptococcus thermophilus.

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

1. the probiotic lactic acid bacteria L.fermentum PM22 and P.acidilactici PM44 are selected from traditional fermented food and are gamma-hemolytic safe strains, and have the characteristics of gastrointestinal condition resistance and high antioxidant activity.

2. The DPPH removing capacity of the fermented milk added with the probiotic starter is remarkably higher than that of a control group (p is less than 0.05), and the antioxidant activity of the fermented milk is remarkably increased by adding the probiotic.

3. The probiotic mixed lactic acid bacteria and the commercial starter are compounded according to a certain proportion, and a symbiotic mechanism is utilized, so that the antioxidant property of the fermented milk is improved, and the adverse effects of fermentation time, post-acidification and the like are reduced.

Drawings

FIG. 1 shows the water holding capacity of fermented milk with mixed lactic acid bacteria in Experimental example 2.

FIG. 2 shows the results of measuring the change in acidity of fermented milk with mixed lactic acid bacteria in Experimental example 3.

FIG. 3 shows the results of measuring the number of viable bacteria in the fermented milk with mixed lactic acid bacteria in Experimental example 5.

Detailed Description

The following examples are further illustrative, but the present invention is not limited to these examples.

Example 1

Taking out the lactobacillus strain frozen in a refrigerator with the temperature of-80 ℃, recovering to the room temperature, inoculating into a sterilized MRS liquid culture medium, and culturing in a biochemical incubator with the constant temperature of 37 ℃ for 24 hours. Activating to 2 generations, compounding according to the compounding proportion designed in the table 1 according to the symbiotic regulation mechanism of the lactic acid bacteria, and putting into a refrigerator at 4 ℃ for storage.

TABLE 1 compounding ratio of lactic acid bacteria

Group of	Bacterial strain	Proportion of addition
			A	L.fermentum PM22:P.pentosaceus PM44	1:2
D	L.fermentum PM22:P.pentosaceus PM44	2:1
			H	L.fermentum PM22:P.pentosaceus PM44	1:1

Example 2

The preparation process of the mixed lactobacillus fermented milk with high antioxidant function in the embodiment is as follows:

1) raw milk: the antibiotics can inhibit the fermentation of the lactic acid bacteria, so that fresh buffalo milk without the antibiotics is selected as raw milk;

2) blending: taking out part of buffalo milk, and adding sucrose accounting for 5 percent of the total mass of the raw milk;

3) homogenizing: heating the buffalo milk obtained in the step 2) to 55 ℃ to dissolve the cane sugar, mixing with the rest of the buffalo milk, stirring uniformly, and homogenizing under the pressure condition of 18Mpa to obtain a base material;

4) and (3) sterilization: sterilizing the base material obtained in the step 3) by adopting a pasteurization mode, heating the base material to 90 ℃, and then preserving heat for 11min to kill pathogenic bacteria and harmful microorganisms in the base material;

5) inoculation: cooling the sterilized base material in the step 4) to 40 ℃, then taking L.fermentum PM22 and P.acidilactici PM44 (volume ratio is 1:2) which are 5% of the total mass of the raw milk, simultaneously adding 0.003% of commercial leavening agent into the cooled base material, stirring until the commercial leavening agent is dissolved and uniformly mixing to obtain inoculated milk;

6) subpackaging and fermenting: subpackaging the obtained inoculated milk, fermenting at constant temperature of 42 ℃, and stopping fermentation when the acidity reaches 70 DEG T to obtain a fermented milk primary product;

7) refrigeration and after-ripening: and (3) refrigerating the fermented milk primary product fermented in the step 6) at the temperature of 2-6 ℃ and then aging for 24 hours.

Example 3

The preparation process of the mixed lactobacillus fermented milk with high antioxidant function in the embodiment is as follows:

1) raw milk: the antibiotic can inhibit the fermentation of the lactic acid bacteria, so that fresh Holstein milk without antibiotic is selected as raw milk;

2) blending: taking out part of the Holstein milk, and adding sucrose accounting for 6 percent of the total mass of the raw milk;

3) homogenizing: heating the holstein milk obtained in the step 2) to 65 ℃ to dissolve the sucrose, mixing with the rest of the holstein milk, stirring uniformly, and homogenizing under the pressure of 19Mpa to obtain a base material;

4) and (3) sterilization: sterilizing the base material obtained in the step 3) by adopting a pasteurization mode, heating the base material to 93 ℃, and then preserving heat for 9min to kill pathogenic bacteria and harmful microorganisms in the base material;

5) inoculation: sterilizing the product obtained in the step 4)Cooling the base material to 45 ℃, then taking the raw milk with 4 percent of L.fermentum PM22 and P.acidilactici PM44 (volume ratio of 2:1) in the total mass, and simultaneously adding 0.006 percent of the raw milk produced by Kehansen LimitedMiId 1.0 is inoculated into the cooled base material, stirred until dissolved and mixed evenly to obtain inoculated milk;

6) subpackaging and fermenting: subpackaging the obtained inoculated milk, fermenting at constant temperature of 38 ℃, and stopping fermentation when the acidity reaches 75 DEG T to obtain a fermented milk primary product;

7) refrigeration and after-ripening: and (3) refrigerating the fermented milk primary product fermented in the step 6) at the temperature of 2-6 ℃ and then aging for 24 hours.

Example 3

The preparation process of the mixed lactobacillus fermented milk with high antioxidant function in the embodiment is as follows:

1) raw milk: the antibiotics can inhibit the fermentation of the lactic acid bacteria, so that fresh goat milk without antibiotics is selected as raw milk;

2) blending: taking out part of goat milk, and adding sucrose accounting for 7% of the total mass of the raw milk;

3) homogenizing: heating the goat milk obtained in the step 2) to 70 ℃ to dissolve the sucrose, mixing with the rest goat milk, stirring uniformly, and homogenizing under the pressure of 20Mpa to obtain a base material;

4) and (3) sterilization: sterilizing the base material obtained in the step 3) by adopting a pasteurization mode, heating the base material to 95 ℃, and then preserving heat for 8min to kill pathogenic bacteria and harmful microorganisms in the base material;

5) inoculation: cooling the sterilized base material in the step 4) to 38 ℃, taking L.fermentum PM22 and P.acidilactici PM44 (volume ratio is 1:1) which account for 3% of the total mass of the raw milk, and simultaneously adding 0.008% of the base material produced by Kehansen LimitedMiId 1.0 is inoculated into the cooled base material, stirred until dissolved and mixed evenly to obtain inoculated milk;

6) subpackaging and fermenting: subpackaging the obtained inoculated milk, fermenting at constant temperature of 38 ℃, and stopping fermentation when the acidity reaches 80 DEG T to obtain a fermented milk primary product;

7) refrigeration and after-ripening: and (3) refrigerating the fermented milk primary product fermented in the step 6) at the temperature of 2-6 ℃ and then aging for 24 hours.

Example 4

The preparation process of the mixed lactobacillus fermented milk with high antioxidant function in the embodiment is as follows:

1) raw milk: the antibiotic can inhibit fermentation of lactobacillus, so fresh Zisan cow milk without antibiotic is selected as raw milk;

2) blending: taking out part of goat milk, and adding sucrose accounting for 6% of the total mass of the raw milk;

3) homogenizing: heating the goat milk obtained in the step 2) to 60 ℃ to dissolve the sucrose, mixing with the rest goat milk, stirring uniformly, and homogenizing under the pressure of 20Mpa to obtain a base material;

4) and (3) sterilization: sterilizing the base material obtained in the step 3) by adopting a pasteurization mode, heating the base material to 92 ℃, and then preserving heat for 10min to kill pathogenic bacteria and harmful microorganisms in the base material;

5) inoculation: cooling the sterilized base material in the step 4) to 40 ℃, taking L.fermentum PM22 and P.acidilactici PM44 (the volume ratio is 1:2) which are 3.5 percent of the total mass of the raw milk, and simultaneously adding 0.005 percent of the base material produced by Kehansen LimitedMiId 1.0 is inoculated into the cooled base material, stirred until dissolved and mixed evenly to obtain inoculated milk;

6) subpackaging and fermenting: subpackaging the obtained inoculated milk, fermenting at constant temperature of 39 ℃, and stopping fermentation when the acidity reaches 80 DEG T to obtain a fermented milk primary product;

7) refrigeration and after-ripening: and (3) refrigerating the fermented milk primary product fermented in the step 6) at the temperature of 2-6 ℃ and then aging for 24 hours.

Comparative example 1

The preparation process of the mixed lactobacillus fermented milk of the embodiment is as follows:

1) raw milk: the antibiotics can inhibit the fermentation of the lactic acid bacteria, so that fresh goat milk without antibiotics is selected as raw milk;

2) blending: taking out part of goat milk, and adding sucrose accounting for 7% of the total mass of the raw milk;

3) homogenizing: heating the goat milk obtained in the step 2) to 70 ℃ to dissolve the sucrose, mixing with the rest goat milk, stirring uniformly, and homogenizing under the pressure of 20Mpa to obtain a base material;

4) and (3) sterilization: sterilizing the base material obtained in the step 3) by adopting a pasteurization mode, heating the base material to 953 ℃, and then keeping the temperature for 8min to kill pathogenic bacteria and harmful microorganisms in the base material;

5) inoculation: cooling the sterilized base material obtained in the step 4) to 38 ℃, and then taking the raw material milk with the total mass of 0.008% produced by Kehansen company LimitedMiId 1.0 is inoculated into the cooled base material, stirred until dissolved and mixed evenly to obtain inoculated milk;

6) subpackaging and fermenting: subpackaging the obtained inoculated milk, fermenting at constant temperature of 38 ℃, and stopping fermentation when the acidity reaches 80 DEG T to obtain a fermented milk primary product;

7) refrigeration and after-ripening: and (3) refrigerating the fermented milk primary product fermented in the step 6) at the temperature of 2-6 ℃ and then aging for 24 hours.

Experimental example 1 measurement of antioxidant Properties of fermented milk with Mixed lactic acid bacteria

The mixed lactic acid bacteria fermented milks having high antioxidant function obtained in example 2 (group a), example 3 (group D) and example 4 (group H) were subjected to examination of effects on antioxidant property, water retention capacity, texture, viable cell count and the like in storage days 1, 4, 7, 10 and 14, and product quality comparison was performed with a control group (comparative example 1) containing no probiotic starter.

Determination of antioxidant Properties of fermented milks:

(1) determination of DPPH radical scavenging Capacity

Diluting sample liquid to be detected by 5 times with ultrapure water, adding 0.5mL of distilled water into 0.5mL of 0.1mmol/LDPPH absolute ethyl alcohol solution, sufficiently shaking, mixing, reacting at room temperature for 30min, and measuring the absorbance at 517nm as A₀Replacing A with the sample liquid to be tested₀The absorbance of the medium distilled water is A₁Replacing A with absolute ethyl alcohol solution₁Absorbance A of the medium DPPH in absolute ethanol₂。

Calculating the formula:

(2) measurement of hydroxyl radical scavenging ability

Sample tube: adding 0.5mL of 1mmol/L phenanthroline absolute ethyl alcohol solution, 1mL of 0.15mol/L phosphate buffer solution with pH of 7.40 and 0.5mL of sample solution to be detected into a 5mL centrifuge tube, mixing uniformly, adding 0.75mmol/L FeSO₄Solution 0.5mL and 0.5mLH₂O₂(volume fraction is 0.01%), mixing, and water bathing at 37 deg.C for 60 min. The absorbance was measured at 536nm as A sample.

Tube damage: and replacing the sample solution to be detected in the sample tube with 0.5mL of distilled water, repeating the operation steps of the sample tube, and measuring the absorbance at 536nm as the loss A.

Undamaged tube: 0.5mL of distilled water was used to replace H in the damaged tube₂O₂. The sample tube procedure was repeated and the absorbance measured at 536nm as A intact

The calculation formula is as follows:

(3) determination of reducing ability

Taking 0.5mL of sample solution to be tested, adding 1.25mL of 0.2mol/L phosphate buffer solution (pH6.6) and 1.25mL of 1% potassium ferricyanide solution, mixing, adding 1.25mL of 10% (W/V) trichloroacetic acid after water bath at 50 ℃ for 20min, mixingStanding at room temperature for 10min after mixing. 1.25mL of distilled water was added to 1.25mL of the supernatant, and 0.1% FeCl was added₃Then mixing evenly, standing for 10min, and measuring the absorbance at 700 nm. And replacing the sample solution to be detected with phosphate buffer solution as a blank control.

TABLE 2 anti-oxidant Properties of fermented milk compounded with Complex lactic acid bacteria and commercial leaven

Group of	Hydroxyl radical scavenging rate (%)	Reducing ability A700	DPPH clearance (%)
				A	90.10±2.69a	15.06±0.00a	0.53±0.03
D	83.46±1.80b	12.27±0.12b	0.52±0.01
				H	75.09±2.83c	12.55±0.51b	0.53±0.02
Control group	53.82±0.51d	8.24±0.53c	0.50±0.04

Note: the different lower case letters in the superscript indicate that there is a significant difference between the same column (p <0.05)

As can be seen from Table 2, the three in vitro antioxidant indices of the three examples are numerically higher than those of the control group. The DPPH clearance of the fermented milk of example 1 is 90.10%, which is significantly higher than that of the other groups (P <0.05), and the same rule is shown in the hydroxyl radical clearance. And the DPPH removing capability of fermented milk added with the probiotic starter is higher than that of a control group (p is less than 0.05). This indicates that the addition of probiotics resulted in a significant increase in the antioxidant activity of the fermented milk.

Experimental example 2 measurement of Water holding Capacity of Mixed lactic acid bacterium fermented milk

Weighing empty centrifuge tube at first and recording the mass as A₀Weighing 20g of fermented milk in a centrifuge tube, recording the mass as m, centrifuging for 20min at 3 ℃ under 6000 Xg, discarding the supernatant, weighing the tube and the precipitate, and recording the mass as A₁The water holding capacity is:

as seen from the results in fig. 1, it can be seen that the water holding capacity of the fermented milks of 4 groups with different leavens was maintained at about 35% during the cold storage, and during the cold storage, the water holding capacity showed a rising trend overall, and after 7 days, the water holding capacity of A, D, H groups was close to or slightly higher than that of the control group. The water holding capacity of the fermented milk is not obviously influenced by adding the probiotic leavening agent.

Experimental example 3 measurement of lactic acid degree Change in fermentation of Mixed lactic acid bacteria

Refer to GB 5009.239-2016.

The results are shown in fig. 2, and it can be seen that the acidity of both the fermented milks of group D and the control group rapidly increased to above 100 ° T during storage for 1-7 days, wherein the titrated acidity of the fermented milks of group D remained at the highest 103.63. Since the change trend of the acidity of the fermented milk of the group D and the group H is more obvious, the fermented milk of the group D and the group H has stronger post-acidification capability compared with the fermented milk of the group A and the group H. After the 10 th day of storage, the 4 groups of fermented milks showed a tendency to decrease. The acidity rise is caused by fermentation of strains to produce acid; the acidity is reduced because the acidity reaches its maximum after fermentation to some extent, and some substances may be generated to neutralize or buffer the acidity and reduce the acidity. In conclusion, the acidity of the 4 groups of fermented milk is between 70-110 DEG T required by GB 5413.34-2010, which indicates that the strains fermented by the 4 groups of fermented milk belong to weak post-acidification strains and can be used for producing fermented milk.

Experimental example 4 measurement of Change in tackiness of fermented milk with Mixed lactic acid bacteria

And (3) taking the sample out of the refrigerator at 4 ℃ and recovering to room temperature, and then carrying out texture analysis by using an FTC professional research grade food physical property analyzer to determine the adhesiveness of the sample. The measurement parameters were as follows: the diameter is 25.60mm, the height is 35.07 mm's cylindrical probe, detection speed is 60mm/min, deformation percentage is 20%, the initial force is 0.05N, the strength response component range is 50N.

The results are shown in Table 3.

TABLE 3 adhesiveness of fermented milk during storage

Group of	Day 1	Day 4	Day 7	Day 10	Day 14
						A	0.21±0.02bc	0.41±0.04a	0.37±0.02a	0.27±0.03b	0.23±0.01b
D	0.32±0.05a	0.40±0.04a	0.41±0.03a	0.37±0.03a	0.37±0.03a
						H	0.25±0.01b	0.33±0.03a	0.19±0.02c	0.33±0.04ab	0.21±0.04b
Control group	0.16±0.01c	0.18±0.01b	0.27±0.02b	0.32±0.03ab	0.24±0.03b

Note: the different lower case letters in the superscript indicate that there is a significant difference between the same column (p <0.05)

From table 3, it can be seen that, in the whole storage process of 14d, the adhesiveness of the three probiotic starter groups in the first 4 days is greater than that of the control group, and the difference between the adhesiveness of the control group and that of the probiotic starter groups is not obvious in the period of 10-14 days, so that the addition of the probiotics is beneficial to improving the adhesiveness of the fermented milk.

Experimental example 5 measurement of viable count of fermented milk with Mixed lactic acid bacteria

The total number of lactic acid bacteria in the fermented milk was measured with reference to GB 4789.35-2016, and the results are shown in FIG. 3.

As is clear from FIG. 3, the number of lactic acid bacteria tended to increase first and then decrease as the storage time extended during the storage at 4 ℃ for 14 days. The change trends of the lactic acid bacteria number in the group A and the group D fermented milks are similar, and both show a descending trend after the 7D reaches the peak value, but the lactic acid bacteria number in the group A fermented milks is always more than that in the group A fermented milks in the storage period. The fact that the group A fermented milk is added with the compound lactobacillus as the auxiliary leavening agent plays a certain role. The number of lactic acid bacteria in the group H fermented milk rapidly increases from 7d to 10d in the storage period, and the number of lactic acid bacteria is 17.7 multiplied by 10⁸CFU/g increased to 22.4X 10⁸CFU/g, gradually decreased after reaching the peak at 10 d. As is clear from FIG. 3, the number of viable bacteria in the 4 groups of fermented milks in this study was 1X 10 in all of the 4 groups of fermented milks in the storage period of 14 days⁸CFU/g or more, viable count maintained above FAO recommended level: (>1×10⁷CFU/g) meets the limit requirement of the number of the lactic acid bacteria in GB 19302-2010. The results show that the compound lactic acid bacteria in the 4 groups of fermented milks have better activity and have the mutual beneficial symbiotic effect with the strains in the commercial leaven, so the compound lactic acid bacteria have the potential of being used as an excellent leaven of the fermented milks.

Experimental example 6 sensory evaluation of fermented milk with Mixed lactic acid bacteria

The evaluation group is composed of 10 food-related professionals, and the sensory evaluation of the yoghourt is carried out from three aspects of color, taste, smell and tissue state, and the full score is 100. The scoring standard is referred to in the literature of Huangli, Li Ling, Yang Pan, and the like, the response surface method optimizes the fermentation process of the polydextrose sour water milk, the food industry, 2016(11): 100-.

TABLE 4 organoleptic evaluation of yoghurts during storage

	Day 1	Day 4	Day 7	Day 10	Day 14
						A	85.17±9.68aA	79.00±3.74bAB	84.50±3.08aA	77.67±4.59aAB	72.00±8.41bB
D	86.17±7.19aA	80.67±5.65bA	81.83±2.71abA	84.33±5.82bA	79.50±8.57abA
						H	84.50±5.61aA	81.83±3.97bA	80.33±1.97abA	82.50±3.51abA	82.00±6.00aA
Control group	78.50±11.27aB	88.00±4.73aA	78.00±6.23bB	82.33±6.12abAB	77.67±6.19abB

Note: lower case letters indicate significant differences between columns (p <0.05), upper case letters indicate significant differences between rows (p <0.05)

As can be seen from table 4, there was no significant difference in the sensory evaluation results of the 4 groups of fermented milks at the 1 st day of the storage period (p >0.05), indicating that the 4 groups of fermented milks did not differ much in color, flavor, odor and texture at the 1 st day of the storage period. The score of the A, D, H fermented milk was significantly lower than that of the control fermented milk by the 4 th d of the storage period, while the score of the a fermented milk was 84.5 significantly higher than that of the 78 of the control fermented milk at the 7 th d of the storage period, indicating that the flavor of the a fermented milk supplemented with the complex lactic acid bacteria as the auxiliary starter during the storage period was more favorable than that of the control fermented milk with the single commercial starter.

With the extension of the 14d storage period, the score 88.00 of the fermented milk in the control group at the 4d storage period is significantly higher than the sensory evaluation score (p <0.05) at the rest storage periods, which indicates that the fermented milk in the control group has the best flavor at the 4d in the whole storage period and is favored by people. And the groups D and H fermented milks have no significant difference in sensory evaluation scores (P >0.05) in the whole storage period, which indicates that the fermented milks have stable flavor and do not generate large flavor changes in the 14D storage period. On the whole, the sensory evaluation score of the fermented milk in the group H is the highest, and is higher than 80 points between 14d storage periods, which shows that the flavor is always better in the 14d storage period and is more favored by people.

Based on the comprehensive evaluation, the addition of the probiotic lactic acid bacteria starter as an auxiliary starter of the commercial starter can improve the antioxidant property and the storage period property of the fermented milk, and is suitable for the production of the fermented milk.