Lactobacillus plantarum Grx403 and application thereof in preparation of fermented enzymolysis skimmed milk

Abstract

The invention discloses a lactobacillus plantarum Grx403 strain and application thereof in preparation of fermented and enzymolyzed skimmed milk. The preservation number of the lactobacillus plantarum Grx403 is CGMCC NO.30468, the lactobacillus plantarum Grx403 can be used for fermenting enzymolysis skimmed milk, the bitterness evaluation value and the taste response value of the enzymolysis skimmed milk are further reduced while the scavenging rate of the enzymolysis skimmed milk to DPPH free radicals and the inhibition rate of the enzymolysis skimmed milk to. OH and SOD are improved, and the lactobacillus plantarum Grx403 has wide application prospects in preparation of lactobacillus food with high antioxidant activity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a plant lactobacillus Grx403 and application thereof in preparing fermented enzymatic defatted milk. Background Art

[0002] Cow's milk can provide essential amino acids, fatty acids, minerals, vitamins, immune-active substances and other nutrients required for human nutrition; however, some protein compounds in cow's milk can easily cause allergic reactions in some people, affecting people's pursuit of healthy food. Enzymatic hydrolysis can not only improve its allergenicity by changing the structure of milk protein, but also give it stronger antioxidant capacity; however, the enzymatic hydrolysis products often have a bitter taste, which affects the sensory perception of the product.

[0003] By fermenting skim milk with lactic acid bacteria and further enzymatically defatting the milk with the aminopeptide enzymes of the lactic acid bacteria themselves, peptides containing hydrophobic amino acid sequences can be decomposed to reduce the bitterness of the skim milk; at the same time, the antioxidant capacity of the skim milk can also be enhanced, which has a good effect on improving its taste and flavor.

[0004] At the same time, as an important probiotic in the intestines of the body, lactic acid bacteria can not only regulate the intestinal flora of the body, but also show strong antioxidant ability in the intestines of the body, promote the production of antioxidant enzymes, and help the body to remove reactive oxygen species (ROS) in the intestines, thereby reducing oxidative damage and benefiting the health of the body. Lactic acid fermentation has become one of the popular ways of food processing. While improving the flavor and quality stability of food, it can also enhance the nutritional value and biological activity of food, achieving the purpose of synergistic enhancement.

[0005] At present, although studies have shown that lactic acid bacteria fermentation can enhance the antioxidant capacity of food and improve its sensory properties such as bitterness and flavor, there is little development of products related to lactic acid bacteria fermentation enzymatic skim milk. Summary of the invention

[0006] The present invention provides a strain of Lactobacillus plantarum Grx403 and its application in preparing fermented enzymatic defatted milk. The present invention uses enzymatic defatted milk with optimized hydrolysis degree and bitterness as a fermentation substrate to screen out lactic acid bacteria with strong antioxidant ability; and uses antioxidant and bitterness as evaluation indicators to optimize the fermentation conditions of lactic acid bacteria fermented enzymatic defatted milk to obtain lactic acid bacteria fermented enzymatic defatted milk with high antioxidant ability, low bitterness and good flavor.

[0007] The present invention provides a strain of Lactobacillus plantarum Grx403, which has the ability to improve the antioxidant capacity of enzymatic skimmed milk and improve its bitterness and flavor. It was deposited in the General Microbiology Center of China National Culture Collection Administration on April 28, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.30468.

[0008] The invention also provides an application of the plant lactobacillus Grx403 in preparing fermented enzymatic skim milk. The plant lactobacillus Grx403 is used to ferment enzymatic skim milk. The preparation method of the enzymatic skim milk is as follows: 12% (W / W) skim milk powder is dissolved in pure water, the pH value is adjusted to 6.5-7.5, and then a composite protease is added, the enzyme amount is 10500-16500 U / g, the enzymatic hydrolysis temperature is 45-55 DEG C, the enzymatic hydrolysis time is 4.5-5h, and after the enzymatic hydrolysis is completed, the enzyme is inactivated and sterilized at 105 DEG C for 5min to obtain the enzymatic skim milk.

[0009] Preferably, the preparation method of enzymatic skim milk is as follows: dissolve 12% (W / W) skim milk powder in pure water, adjust the pH to 6.5, then add composite protease, the enzyme amount is 13500U / g, the enzymatic hydrolysis temperature is 55°C, the enzymatic hydrolysis time is 4.5h, and after the enzymatic hydrolysis is completed, sterilize at 105°C for 5min to obtain enzymatic skim milk.

[0010] Preferably, the pH is adjusted using a 0.1 mol / L NaOH solution.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] (1) The present invention uses the degree of hydrolysis, bitterness evaluation value and taste response value as evaluation indicators to screen the types of enzymes, and optimizes the conditions for enzymatic defatting of milk, thereby obtaining a composite protease enzymatic defatting milk with a higher degree of hydrolysis, lower bitterness evaluation value and taste response value, and uses this as a substrate for strain fermentation.

[0013] (2) The present invention inoculates a lactic acid bacteria strain into an optimized composite protease enzymatic skim milk, provides a method for measuring the scavenging rate of the enzymatic skim milk on DPPH free radicals, and the inhibition rate of ·OH and SOD, thereby obtaining a lactic acid bacteria strain capable of improving the antioxidant capacity of the enzymatic skim milk, and further tests the bitterness evaluation value, taste response value and volatile flavor substances of the lactic acid bacteria strain capable of improving the antioxidant capacity of the enzymatic skim milk, thereby obtaining a plant lactobacillus Grx403 capable of improving the antioxidant capacity of the enzymatic skim milk and improving its bitterness and flavor.

[0014] (3) The plant lactobacillus Grx403 of the present invention improves the scavenging rate of enzymatic skimmed milk on DPPH free radicals and the inhibition rate of ·OH and SOD, while further reducing the bitterness evaluation value and taste response value of the enzymatic skimmed milk and improving its flavor. The fermented enzymatic skimmed milk has a higher scavenging rate for DPPH free radicals, a higher inhibition rate for ·OH and SOD, and a lower bitterness. It has broad application prospects in the preparation of lactic acid bacteria foods with high antioxidant activity, such as lactic acid bacteria solid beverages, postbiotics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the hydrolysis degree of skim milk enzymatically processed by three kinds of proteases, among which A is skim milk enzymatically processed by neutral protease; B is skim milk enzymatically processed by flavor protease; C is skim milk enzymatically processed by compound protease; different letters indicate that the data are significantly different (p<0.05);

[0016] Figure 2 The bitterness of three kinds of skim milk enzymatically processed by proteases, A is skim milk enzymatically processed by neutral protease; B is skim milk enzymatically processed by flavor protease; C is skim milk enzymatically processed by compound protease; different letters indicate that the data are significantly different (p<0.05);

[0017] Figure 3 is the effect of quinine concentration on taste response value;

[0018] Figure 4 The electronic tongue taste of enzymatic skim milk;

[0019] Figure 5 The effect of time on the hydrolysis degree of enzymatic skim milk, where different letters indicate significant differences (p<0.05);

[0020] Figure 6 The effect of enzyme addition on the hydrolysis degree of enzymatic skim milk, where different letters indicate significant differences in the data (p<0.05);

[0021] Figure 7 The effect of temperature on the hydrolysis degree of enzymatic skim milk, where different letters indicate significant differences in the data (p<0.05);

[0022] Figure 8 The effect of skim milk pH on the hydrolysis degree of enzymatic skim milk, where different letters indicate significant differences in the data (p<0.05);

[0023] Fig. 9are the bitterness evaluation values ​​of enzymatic skimmed milk and enzymatic skimmed milk fermented by four lactic acid bacteria, among which A is the bitterness evaluation value of enzymatic skimmed milk; B is the sample of enzymatic skimmed milk fermented by Lactococcus lactis m16; C is the sample of enzymatic skimmed milk fermented by Lactobacillus plantarum 97; D is the sample of enzymatic skimmed milk fermented by Lactobacillus plantarum Grx403; E is the sample of enzymatic skimmed milk fermented by Lactococcus lactis g21; different letters indicate that the data are significantly different (p<0.05);

[0024] Fig.10 The electronic tongue taste of skim milk fermented with lactic acid bacteria enzymes;

[0025] Fig.11 is a morphological diagram of Lactobacillus plantarum Grx403;

[0026] Fig.12 This is the electrophoresis diagram of the 16S rDNA PCR amplification product of Lactobacillus plantarum Grx403;

[0027] Fig.13 The antioxidant capacity of skimmed milk fermented by Lactobacillus plantarum Grx403 at different fermentation temperatures, where A is the effect of fermentation temperature on DPPH free radical scavenging rate; B is the effect of fermentation temperature on ·OH inhibition rate; C is the effect of fermentation temperature on SOD inhibition rate; different letters indicate that the data are significantly different (p<0.05);

[0028] Fig.14 The antioxidant capacity of skimmed milk fermented by Lactobacillus plantarum Grx403 at different fermentation times, where A is the effect of fermentation time on DPPH free radical scavenging rate; B is the effect of fermentation time on ·OH inhibition rate; C is the effect of fermentation time on SOD inhibition rate; different letters indicate that the data are significantly different (p<0.05);

[0029] Fig.15 The antioxidant capacity of skimmed milk fermented by Lactobacillus plantarum Grx403 at different inoculation amounts, where A is the effect of inoculation amount on DPPH free radical scavenging rate; B is the effect of inoculation amount on ·OH inhibition rate; C is the effect of inoculation amount on SOD inhibition rate; different letters indicate that the data are significantly different (p<0.05);

[0030] Fig.16 The antioxidant capacity of skim milk fermented by Lactobacillus plantarum Grx403 at different skim milk pH, where A is the effect of skim milk pH on DPPH free radical scavenging rate; B is the effect of skim milk pH on ·OH inhibition rate; C is the effect of skim milk pH on SOD inhibition rate; different letters indicate that the data are significantly different (p<0.05);

[0031] Fig.17Response surface diagram and contour diagram of the interaction effect on the comprehensive evaluation value. DETAILED DESCRIPTION

[0032] The present invention is further described below by specific examples and drawings, but the present invention is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product specifications.

[0033] In the following examples, the neutral protease (Neutrase 0.8L), flavor protease (Flavourzyme 1000L), and composite protease (Protamex 1.6) used were provided by Novozymes (China) Biotechnology Co., Ltd.

[0034] Example 1 Preparation of enzymatic skim milk

[0035] In this example, a composite protease was selected for enzymatic hydrolysis through tests of degree of hydrolysis, bitterness evaluation value and electronic tongue taste response value, and the enzymatic hydrolysis time, enzyme addition amount, enzymatic hydrolysis temperature and skim milk pH were optimized with the degree of hydrolysis as the evaluation index, thereby producing enzymatic skim milk with high degree of hydrolysis and low bitterness.

[0036] 1. Effect of protease on hydrolysis degree and bitterness of skim milk

[0037] Prepare a 12% (W / W) skim milk solution with purified water, adjust the temperature and pH of the skim milk to the optimal conditions for each protease using a water bath and 0.5 mol / L food-grade sodium hydroxide, and take samples after 4.5 hours of enzymolysis. After the enzymolysis is completed, put the enzymatic skim milk into a sterilizer at 105°C for 5 minutes to inactivate the enzyme, and store it in a refrigerator at 4°C for subsequent analysis. The enzymolysis conditions of neutral protease, flavor protease and composite protease are shown in Table 1.

[0038] Table 1 Enzymatic hydrolysis conditions of protease

[0039]

[0040] (1) Effect of protease on the hydrolysis degree of skim milk

[0041] The degree of hydrolysis (DH) of enzymatic skim milk was determined by pH-Stat method. Take 100 mL of enzymatic skim milk, record the volume B of 0.5 mol / L food-grade sodium hydroxide required to adjust to the optimum pH value each time, and calculate the degree of hydrolysis using formula (1):

[0042]

[0043] Where: B is the volume of food-grade sodium hydroxide solution, mL; M bis the concentration of NaOH solution, which is 0.5 mol / L; α is the average dissociation degree of amino group, 1 / α is 2.26; M p is the mass of protein, which is 3.84 g; h tot For casein, the value is 8.2 mmol / g.

[0044] The degree of hydrolysis is an important parameter to measure the degree of enzymatic hydrolysis, which affects the solubility of protein and the clarity of the enzymatic hydrolysate. Figure 1 .

[0045] Depend on Figure 1 It can be seen that the hydrolysis degree of skim milk enzymatically hydrolyzed by the composite protease is 28.13%, which is significantly higher than 24.89% of neutral protease and 23.20% of flavor protease (p<0.05), indicating that the hydrolysis degree of skim milk hydrolyzed by the composite protease is higher.

[0046] (2) Effect of protease on the bitterness evaluation value of skim milk

[0047] The protein in the enzymatic skim milk will produce hydrophobic peptides, which will produce bitter taste after contacting the taste receptors on the taste buds. -5 , 1×10 -4 , 2×10 -4 , 4×10 -4 , 8×10 -4 and 1.6×10 -3 g / mL quinine standard solution, the corresponding scores are 0, 2, 4, 6, 8 and 10 points. The lower the score, the less bitter it is. A total of 10 trained food graduate students and teachers participated in the evaluation, including 5 males and 5 females, and scored the samples according to the standard scores. The effects of the three proteases on the bitterness of enzymatic skim milk are shown in Figure 2 .

[0048] Depend on Figure 2 It can be seen that the bitterness evaluation value of the skimmed milk enzymatically processed by neutral protease was 7.60, which was significantly higher than 6.80 of compound protease and 6.60 of flavor protease (p<0.05), indicating that the bitterness of skimmed milk enzymatically processed by flavor protease and compound protease was lower.

[0049] (3) Effect of protease on the taste response value of skim milk

[0050] The taste response values ​​of enzymatic skimmed milk were measured using SA402B electronic tongue. The results were as follows: Figure 3 and Figure 4 shown.

[0051] Depend on Figure 3It can be seen that the taste response value of quinine will gradually increase with the increase of its concentration, and the concentration of flavor substances and the flavor intensity show an S-shaped relationship. -4 g / mL, the growth rate of the taste response value is higher than the growth rate of the concentration, which is the "expansion stage"; when the quinine concentration is 4×10 -4 Up to 8×10 -4 g / mL, there was a linear relationship between the taste response value and the concentration; when the quinine concentration exceeded 8×10 -4 g / mL, the growth of the taste response value tends to be stable and no longer changes significantly. The relationship between the response value and concentration at this stage can be expressed by the logarithmic function y = 3.84 × ln (x) + 31.55 (R 2 =0.9212) to fit. Through this function, we can more accurately calculate the quinine equivalent value corresponding to the sample within a specific taste response value range.

[0052] In the electronic tongue sensor analysis, the taste response value expresses the instantaneous strongest bitterness intensity, and the aftertaste-B response value reflects the continuous intensity of bitterness. Therefore, the present invention uses two taste indicators, bitterness and aftertaste-B, to measure the bitterness intensity of enzymatic skim milk. The results are shown in Figure 4 .

[0053] Depend on Figure 4 It can be seen that the taste response value of the composite protease is 5.73, which is lower than 5.97 of the flavor protease and 6.23 of the neutral protease; from the analysis of the aftertaste-B response value results, the aftertaste-B response value of the flavor protease is 5.76, which is lower than 6.06 of the composite protease and 6.11 of the neutral protease.

[0054] joint Figure 3 and Figure 4 The taste response values ​​of neutral protease, flavor protease and compound protease were between 5 and 7, and the quinine concentration was 4×10 -4 -8×10 -4 g / mL, and the corresponding bitterness evaluation value was in the range of 6-8 points, indicating that the sensory evaluation was consistent with the electronic tongue result, proving that the combined application of artificial sensory evaluation and intelligent sensory evaluation methods in the bitterness evaluation of enzymatic skimmed milk was reliable.

[0055] Based on the hydrolysis degree, bitterness sensory evaluation and electronic tongue taste results of enzymatic skim milk, it was found that the hydrolysis degree of enzymatic skim milk with composite protease was higher and the bitterness was lower.

[0056] 2. Optimization of skim milk enzymatic hydrolysis conditions - single factor experiment

[0057] (1) Effect of enzymatic hydrolysis time on the hydrolysis degree of skim milk by compound protease

[0058] The enzymatic hydrolysis temperature was 50°C, the skim milk pH was 7.0, the enzyme addition amount was 10500U / g, and the enzymatic hydrolysis time was 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, and 7.5h, respectively. The results are shown in Figure 5 .

[0059] Depend on Figure 5 It can be seen that the hydrolysis degree of enzymatic skim milk increases first and then stabilizes with the enzymatic hydrolysis time. When the enzymatic hydrolysis time is 4.5h, the hydrolysis degree of composite protease enzymatic skim milk reaches a maximum of 28.13%, which is significantly higher than the hydrolysis degree of 25.19% at 3.5h (p<0.05).

[0060] (2) Effect of enzyme addition amount on the hydrolysis degree of skim milk by composite protease

[0061] The enzymatic hydrolysis temperature was 50°C, the skim milk pH was 7.0, the enzymatic hydrolysis time was 4.5 h, and the enzyme addition amounts were 1500, 4500, 7500, 10500, 13500, and 16500 U / g. The results are shown in Figure 6 .

[0062] Depend on Figure 6 It can be seen that the degree of hydrolysis first increases and then decreases with the increase of enzyme addition. When the enzyme addition amount is 13500U / g, the degree of hydrolysis of skim milk enzymatically prepared by composite protease reaches a maximum value of 29.96%, which is significantly higher than the hydrolysis degree of 28.34% at 10500U / g and the hydrolysis degree of 26.09% at 16500U / g (p<0.05).

[0063] (3) Effect of enzymatic hydrolysis temperature on the hydrolysis degree of skim milk by composite protease

[0064] The pH of skim milk was 7.0, the enzyme addition amount was 10500U / g, the enzymatic hydrolysis time was 4.5h, and the enzymatic hydrolysis temperatures were 45℃, 50℃, 55℃, 60℃, and 65℃, respectively. The results are shown in Figure 7 .

[0065] Depend on Figure 7 It can be seen that the hydrolysis degree of enzymatic skim milk first increases and then decreases with the increase of enzymatic hydrolysis temperature. When the enzymatic hydrolysis temperature is 50℃, the hydrolysis degree of composite protease enzymatic skim milk reaches a maximum of 28.36%, which is significantly higher than the hydrolysis degree of 27.37% at the enzymatic hydrolysis temperature of 45℃ and the hydrolysis degree of 27.04% at the enzymatic hydrolysis temperature of 55℃ (p<0.05).

[0066] (4) Effect of skim milk pH on the hydrolysis degree of skim milk by composite protease

[0067] The enzymatic hydrolysis temperature was 50°C, the enzyme addition amount was 10500U / g, the enzymatic hydrolysis time was 4.5h, and the skim milk pH was 6.5, 7.0, 7.5, 8.0, and 8.5, respectively. The results are shown in Figure 8 .

[0068] Depend on Figure 8 It can be seen that the hydrolysis degree of skim milk with enzymatic enzymes first increased and then decreased with the increase of skim milk pH. At skim milk pH 7.0, the hydrolysis degree of skim milk with composite protease reached a maximum of 28.74%, which was significantly higher than the hydrolysis degree of 21.66% at skim milk pH 6.5 and 25.52% at pH 7.5 (p<0.05).

[0069] 3. Orthogonal experimental optimization of skim milk enzymatic hydrolysis conditions

[0070] Combined with the results of the single factor test, orthogonal test optimization was carried out with enzymatic hydrolysis temperature, enzyme addition amount and skim milk pH as independent variables. The specific parameters are shown in Table 2, and the orthogonal test results are shown in Table 3.

[0071] Table 2 Conditional factor levels of compound protease enzymatic skim milk

[0072]

[0073] Table 3 Results of orthogonal test of compound protease enzymatic skim milk conditions

[0074]

[0075] As shown in Table 3, the order of parameters affecting enzymatic skim milk (i.e., the order of R value) is: enzyme addition > skim milk pH > enzymatic hydrolysis temperature; the optimal parameter combination for enzymatic skim milk is A3B2C1, that is, the parameter values ​​are: using composite protease, temperature 55°C, enzyme addition 13500U / g, pH 6.5. The optimal parameter combination was used for verification, and the hydrolysis degree of composite protease enzymatic skim milk was 43.69%, which is consistent with the orthogonal test results, indicating that the parameters optimized using this model are reliable.

[0076] Example 2 Screening of lactic acid bacteria strains for enhancing the antioxidant capacity of enzymatic skim milk

[0077] In this example, enzymatic skim milk with low bitterness and high hydrolysis degree was used as substrate, and the lactic acid bacteria strains capable of enhancing the antioxidant capacity of enzymatic skim milk were screened out by DPPH free radical scavenging rate, ·OH inhibition rate and SOD inhibition rate.

[0078] 1. Activation and subculture of strains

[0079] 46 lactic acid bacteria strains were inoculated into MRS liquid culture medium at an inoculation rate of 3% (V / V), and cultured in a 37° C. incubator for 18 h for later use.

[0080] 2. Preparation of enzymatic skim milk

[0081] Dissolve 12% (W / W) skim milk powder in 50°C pure water, adjust the pH to 6.5 with 0.1 mol / L NaOH, then add composite protease at an enzyme dosage of 13500 U / g, enzymolysis temperature of 55°C, enzymolysis time of 4.5 h, sterilize at 105°C for 5 min, and cool for use.

[0082] 3. Determination of the antioxidant capacity of skimmed milk fermented by lactic acid bacteria

[0083] The activated lactic acid bacteria strains were centrifuged at 8000×g for 10 min, the cells were collected, washed twice with sterile water, and the concentration was adjusted to 1×10 9 The 3% inoculum was inoculated into enzymatic skim milk with a pH of 6.5 and cultured at 37°C for 18 h. The DPPH free radical scavenging rate, ·OH inhibition rate and SOD inhibition rate were determined according to formulas (2), (3) and (4), respectively. The results are shown in Table 4.

[0084]

[0085] Table 4 Antioxidant capacity of skimmed milk fermented with lactic acid bacteria (n=3, x±sd)

[0086]

[0087]

[0088] Note: In the same column, different letters indicate significant differences (p<0.05)

[0089] As shown in Table 4, the DPPH free radical scavenging rates of enzymatic skim milk fermented by Lactococcus lactis g21, Lactobacillus plantarum Grx403 and Lactobacillus plantarum 97 were significantly higher than those of other strains (p<0.05), which were 63.54%, 57.91% and 57.55% respectively; the ·OH inhibition rates of enzymatic skim milk fermented by Lactococcus lactis m16, Lactobacillus plantarum Grx403 and Lactobacillus fermentum B-74-M17-1-2 were significantly higher than those of other strains (p<0.05), which were 81.72%, 81.58% and 81.25% respectively; the SOD inhibition rates of enzymatic skim milk fermented by Lactococcus lactis g21, Lactococcus lactis m16 and Lactobacillus plantarum Grx403 were significantly higher than those of other strains (p<0.05), which were 69.05%, 60.33% and 59.45% respectively. After comprehensive consideration, Lactococcus lactis g21 and m16, and Lactobacillus plantarum Grx403 and 97 were selected for subsequent experiments.

[0090] Example 3 Effect of lactic acid bacteria on the bitterness and flavor of fermented enzymatic skim milk

[0091] This example studies the effects of Lactococcus lactis g21, Lactococcus lactis m16, Lactobacillus plantarum Grx403 and Lactobacillus plantarum 97 with strong antioxidant capacity on the bitterness and volatile flavor substances of enzymatic skim milk through bitterness sensory evaluation, electronic tongue, GCMS and other tests, and provides candidate strains for the development of lactic acid bacteria fermented enzymatic skim milk products. The preparation of lactic acid bacteria fermented enzymatic skim milk is the same as in Example 2.

[0092] 1. Determination of bitterness of skimmed milk fermented by lactic acid bacteria

[0093] The bitterness evaluation value and taste response value of four lactic acid bacteria fermented enzymatic defatted milk were measured by bitterness sensory evaluation and SA402B electronic tongue instrument, and the results are shown in Fig. 9 and Fig.10 .

[0094] Depend on Fig. 9 It can be seen that the bitterness evaluation values ​​of enzymatic skimmed milk fermented by Lactobacillus plantarum Grx403 and 97 were 4.80 and 5.05, respectively, which were significantly lower than 6.15 and 6.10 of Lactococcus lactis m16 and g21 (p<0.05).

[0095] Depend on Fig.10 It can be seen that the taste response values ​​of Lactobacillus plantarum Grx403 and 97 fermented enzymatic skim milk were 5.10 and 5.21, respectively, which were significantly lower than 7.80 and 7.77 of Lactococcus lactis g21 and m16 (p<0.05), indicating that Lactobacillus plantarum Grx403 and 97 have a good effect on improving the bitterness of enzymatic skim milk.

[0096] 2. Determination of volatile flavor substances in skimmed milk fermented by lactic acid bacteria

[0097] The relative contents of volatile flavor substances in enzymatic defatted milk fermented by four lactic acid bacteria were determined by GCMS. The results are shown in Table 5.

[0098] Table 5 Effect of lactic acid bacteria fermentation on volatile flavor substances in enzymatic skim milk

[0099]

[0100]

[0101]

[0102] As shown in Table 5, the fermentation of the four lactic acid bacteria changed the volatile substances and their contents in the enzymatic skim milk. The fermentation of the four lactic acid bacteria reduced the relative content of aldehydes in the enzymatic skim milk, and aldehydes contribute more to the fishy smell and produce unpleasant odors, such as benzaldehyde with almond and wood flavors, nonanal with butter flavor, and decanal with waxy flavor; the relative content of alcohols and ketones in the enzymatic skim milk was increased, and alcohols and ketones contribute more to the aroma, such as sweet and slightly grassy octanol, mushroom and grassy 1-octen-3-ol, and green apple aroma isopentanol, and ketones mainly present fat flavor, enriching the overall flavor. Among them, compared with the other three strains, Lactobacillus plantarum Grx403 effectively reduced the relative content of aldehydes such as decanal, natural nonanal and benzaldehyde in enzymatic defatted milk, and the overall relative content of aldehydes was also lower than that of the other three strains; it also increased the relative content of alcohols such as n-hexanol and isopentanol in enzymatic defatted milk, and the overall relative content of alcohols was higher than that of the other three strains.

[0103] Therefore, the Lactobacillus plantarum Grx403 of the present invention can not only improve the antioxidant capacity of enzymatic skim milk, but also effectively reduce its bitterness and improve its flavor.

[0104] Example 4 Identification of strain Grx403

[0105] 1. Strain API identification

[0106] The strain Grx403 was identified according to the operating instructions of the API 50CHL series identification reagent strips. The results are shown in Tables 6 and 7.

[0107] Table 6 API 50CHL system identification results

[0108]

[0109] Note: “+” indicates positive; “-” indicates negative

[0110] Table 7 API comparison results

[0111]

[0112] It can be seen from Table 6 and Table 7 that the strain Grx403 of the present invention is Lactobacillus plantarum, and the identification rate is 99%.

[0113] 2. Identification of strains by 16S rDNA sequencing

[0114] The genomic DNA of strain Grx403 (nucleotide sequence of 16S rDNA is shown in SEQ ID NO. 1) was used as a template for PCR amplification, and universal 16S rDNA primers were used for PCR amplification; after electrophoresis detection of the amplified product, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence obtained by sequencing was submitted to BLAST for comparison analysis, and the results are shown in Table 8.

[0115] Table 8 16S rDNA comparison results

[0116]

[0117] As shown in Table 8, strain Grx403 is Lactobacillus plantarum with a homology of 99%. Combined with the comparison results of API50CHL and 16S rDNA sequence, strain Grx403 of the present invention was identified as Lactobacillus plantarum.

[0118] Example 5 Optimization of fermentation conditions for enzymatic skim milk fermentation with Lactobacillus plantarum Grx403

[0119] This example uses a single factor test to investigate the effects of inoculation amount, fermentation temperature, fermentation time and pH of enzymatic skim milk on the DPPH free radical scavenging rate, OH inhibition rate and SOD inhibition rate of enzymatic skim milk fermented by lactic acid bacteria, determine the optimal fermentation condition range, and then optimize the fermentation conditions of enzymatic skim milk fermented by lactic acid bacteria through response surface experiment. The preparation of enzymatic skim milk is the same as that of Example 2.

[0120] 1. Single factor test

[0121] (1) Effect of fermentation temperature on the antioxidant capacity of skim milk fermented by Lactobacillus plantarum Grx403

[0122] The fermentation time was 18.0h, the inoculum amount of Lactobacillus plantarum Grx403 was 3.0%, and the pH of the enzymatic skim milk was 6.5; the fermentation temperatures were 25.0℃, 29.0℃, 33.0℃, 37.0℃, 41.0℃, and 45.0℃, respectively. The results are shown in Fig.13 .

[0123] Depend on Fig.13It can be seen that when the fermentation temperature is 37.0℃, the DPPH free radical scavenging rate reaches the highest, which is 57.00%, followed by 33.0℃ and 41.0℃, with scavenging rates of 53.86% and 52.36% respectively; when the fermentation temperature is 33.0℃, the ·OH inhibition rate reaches the highest, which is 85.62%, followed by 29.0℃ and 37.0℃, with inhibition rates of 77.94% and 81.65% respectively; at 33.0℃, the SOD inhibition rate also reaches the highest, which is 60.86%, followed by 29.0℃ and 37.0℃, with inhibition rates of 58.61% and 59.00% respectively. Comprehensively considering the antioxidant capacity results and further refining the effect of temperature on antioxidant capacity, the fermentation temperatures of 33.0℃, 35.0℃ and 37.0℃ were selected for subsequent response surface experiments.

[0124] (2) Effect of fermentation time on the antioxidant capacity of skim milk fermented by Lactobacillus plantarum Grx403

[0125] The fermentation temperature was 37.0℃, the inoculum amount of Lactobacillus plantarum Grx403 was 3.0%, and the pH of the enzymatic skim milk was 6.5; the fermentation time was 10.0, 12.0, 14.0, 16.0, 18.0, and 22.0h, respectively. The results are shown in Fig.14 .

[0126] Depend on Fig.14 It can be seen that when the fermentation time was 16.0h, the DPPH free radical scavenging rate reached the highest, which was 59.10%, followed by 14.0h and 18.0h, with scavenging rates of 57.22% and 57.74% respectively; when the fermentation time was 16.0h, the ·OH inhibition rate reached the highest, which was 84.59%, followed by 14.0h and 18.0h, with inhibition rates of 75.94% and 80.01% respectively; when the fermentation time was 16.0h, the SOD inhibition rate reached the highest, which was 62.10%, followed by 14.0h and 18.0h, with inhibition rates of 61.22% and 59.74% respectively. Based on the antioxidant capacity results, the fermentation times of 14.0, 16.0 and 18.0h were selected for subsequent response surface experiments.

[0127] (3) Effect of inoculum dose on antioxidant capacity of enzymatic skim milk fermented by Lactobacillus plantarum Grx403

[0128] The fermentation temperature was 37.0℃, the fermentation time was 18.0h, the enzymatic skim milk pH was 6.5; the inoculation amount of Lactobacillus plantarum Grx403 was 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, and 6.0%, respectively. The results are shown in Fig.15 .

[0129] Depend on Fig.15It can be seen that when the inoculation amount was 4.0%, the DPPH free radical scavenging rate reached the highest, which was 64.26%, followed by 3.0% and 5.0%, with scavenging rates of 57.00% and 59.20% respectively; when the inoculation amount was 4.0%, the ·OH inhibition rate reached the highest, which was 82.17%, followed by 3.0% and 5.0%, with scavenging rates of 80.35% and 80.14% respectively; when the inoculation amount was 3.0%, the SOD inhibition rate reached the highest, which was 59.42%, followed by 2.0% and 4.0%, with inhibition rates of 49.25% and 54.18% respectively. Based on the antioxidant capacity results, the inoculation amounts of 3.0%, 4.0%, and 5.0% were selected for subsequent response surface experiments.

[0130] (4) Effect of pH of enzymatic skim milk on the antioxidant capacity of enzymatic skim milk fermented by Lactobacillus plantarum Grx403

[0131] The fermentation temperature was 37.0℃, the fermentation time was 18.0h, the inoculum amount of Lactobacillus plantarum Grx403 was 3.0%; the pH of the enzymatic skim milk was 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, respectively. Fig.16 .

[0132] Depend on Fig.16 It can be seen that when the pH of the enzymatic skim milk was 6.0, the DPPH free radical scavenging rate reached the highest, which was 60.22%, followed by pH 5.5 and pH 6.5, with scavenging rates of 59.42% and 57.55% respectively; when the pH of the enzymatic skim milk was 5.5, the ·OH inhibition rate reached the highest, which was 84.88%, followed by pH 5.0 and pH 6.0, with inhibition rates of 83.55% and 83.41% respectively; when the pH of the enzymatic skim milk was 6.5, the SOD inhibition rate reached the highest, which was 59.55%, followed by pH 6.0 and pH 7.0, with inhibition rates of 58.84% and 54.73% respectively. Based on the antioxidant capacity results, the pH of the enzymatic skim milk was selected to be 5.5, 6.0, and 6.5 for subsequent response surface experiments.

[0133] 2. Response surface experiment

[0134] The appropriate level range of each factor was selected, and the four factors of Lactobacillus plantarum Grx403 inoculation amount, fermentation temperature, fermentation time and pH of enzymatic skim milk were taken as independent variables. The fuzzy comprehensive evaluation value of bitterness sensory evaluation of fermented enzymatic skim milk, DPPH free radical scavenging rate, ·OH inhibition rate and SOD inhibition rate were taken as response values. The fermentation conditions of Lactobacillus plantarum Grx403 fermented enzymatic skim milk were optimized through response surface experiment. The factor level design of response surface experiment is shown in Table 9, the results are shown in Table 10 and Table 11, and the interaction and contour lines of response surface are shown in Table 12. Fig.17 .

[0135] Table 9 Factor level table

[0136]

[0137] Table 10 Response surface experimental design scheme

[0138]

[0139] Table 11 Results of variance analysis of response surface experiment

[0140]

[0141] The data were analyzed using Design-Expert 11 software, and the quadratic polynomial regression equation was obtained as follows: Y = 0.5009 + 0.0761A + 0.0193B - 0.0075C + 0.0263D - 0.0502AB - 0.0241AC + 0.0505AD + 0.0141BC + 0.0250BD - 0.0224CD + 0.0579A 2 -0.0181B 2 -0.0436C 2 +0.0307D 2 , where A is the fermentation time, B is the fermentation temperature, C is the inoculum amount, D is the pH of the enzymatic skimmed milk, and Y is the fuzzy comprehensive evaluation value.

[0142] As shown in Table 11, the F value of the model is 12.75 (p < 0.05), which is statistically significant, and the lack of fit term is not significant (p = 0.1665 > 0.05), which proves that the mathematical model is appropriate. 2 =0.9273, Corrected determination coefficient R 2 Adj = 0.8545, indicating that the equation has a good fitting degree, and it is feasible to use this model to predict and determine the optimal process conditions. According to the size of the P value, it can be reflected that the contribution rate of each factor to the response value is A>D>B>C, that is, fermentation temperature>enzyme skim milk pH>fermentation time>inoculation amount.

[0143] Depend on Fig.17 It can be seen that there is an obvious slope change between fermentation time and inoculation amount, and the contour shape is similar to an ellipse, which reflects that the interaction between the two has a significant effect on the fuzzy comprehensive evaluation value of Lactobacillus plantarum Grx403 in the process of fermenting enzymatic skim milk (pAB<0.05).

[0144] Considering the actual situation, the optimal fermentation conditions for Lactobacillus plantarum Grx403 fermented enzymatic skimmed milk were obtained as follows: fermentation temperature 37 ° C, fermentation time 18 h, Lactobacillus plantarum Grx403 inoculation amount 5%, enzymatic skimmed milk pH 6.5, and the predicted fuzzy comprehensive evaluation value was 0.741. Under these conditions, the sensory evaluation of Lactobacillus plantarum Grx403 fermented enzymatic skimmed milk was 4.25, the DPPH free radical scavenging rate was 61.32%, the ·OH inhibition rate was 83.35%, and the SOD inhibition rate was 60.77%.

[0145] Example 6 Preparation of lactic acid bacteria solid beverage with high antioxidant activity

[0146] The preparation of enzymatic skim milk is the same as that of Example 2. 5% Lactobacillus plantarum Grx403 is inoculated into enzymatic skim milk with a pH of 6.5, and fermented and cultured at 37°C for 18 hours, and a protective agent of Lactobacillus plantarum Grx403 is added, and a freeze-dried powder with a water content of not more than 3% is obtained after vacuum freeze drying, and the freeze-dried powder is mixed with maltodextrin and oligoxylose in a certain proportion under aseptic conditions to obtain a Lactobacillus plantarum Grx403 solid beverage.

[0147] Example 7 Preparation of postbiotic products stored at room temperature

[0148] The preparation of enzymatic skim milk is the same as that of Example 2. 5% Lactobacillus plantarum Grx403 was inoculated into enzymatic skim milk with a pH of 6.5, and fermented and cultured at 37°C for 18 hours. After pasteurization, the milk was crushed by aseptic high-pressure homogenization to further release the active substances in the probiotics, thus obtaining a postbiotic product with high antioxidant activity.

Claims

1. Lactobacillus plantarum ( Lactobacillus plantarum ) Grx403, characterized in that, The deposit number is CGMCCNO. 30468.

2. The use of Lactobacillus plantarum Grx403 as claimed in claim 1 in preparing fermented enzymatic skim milk, characterized in that: Enzymatic skim milk is fermented by using the plant lactobacillus Grx403 described in claim 1. The preparation method of the enzymatic skim milk is as follows: 12% (W / W) skim milk powder is dissolved in pure water, the pH is adjusted to 6.5-7.5, and then a composite protease is added, the enzyme amount is 10500-16500 U / g, the enzymolysis temperature is 45-55°C, the enzymolysis time is 4.5-5 h, and after the enzymolysis is completed, the enzyme is inactivated and sterilized at 105°C for 5 min to obtain the enzymatic skim milk.

3. The use according to claim 2, characterized in that: The preparation method of the enzymatic skim milk is as follows: 12% (W / W) skim milk powder is dissolved in pure water, the pH is adjusted to 6.5, and then a composite protease is added, the enzyme amount is 13500 U / g, the enzymatic hydrolysis temperature is 55°C, the enzymatic hydrolysis time is 4.5 h, and after the enzymatic hydrolysis is completed, the enzyme is inactivated and sterilized at 105°C for 5 min to obtain the enzymatic skim milk.

4. The use according to claim 2 or 3, characterized in that: The pH was adjusted using 0.1 mol / L NaOH solution.

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