A probiotic sea cucumber peptide powder that helps improve immunity and its preparation method

Abstract

The present invention provides a probiotic sea cucumber peptide powder that helps to improve immunity and a preparation method thereof, belonging to the technical field of food processing. The present invention comprehensively uses the method of stepwise enzymatic hydrolysis with papain and neutral protease, and fermentation with Eurotium cristatum and / or Bacillus coagulans to prepare a deodorized probiotic sea cucumber peptide powder. The method of the present invention does not require enzyme inactivation and decolorization treatment during the whole preparation process. The probiotic sea cucumber peptide powder obtained by this method has no fishy smell, is light yellow in color and has a smooth luster, and has a high viable count of probiotics. It is suitable for the daily health care of people with low immunity.

Description

Technical Field

[0001] The present invention relates to the field of aquatic product processing, and particularly relates to a probiotic sea cucumber peptide powder helpful for improving immunity and a preparation method thereof. Background Art

[0002] Since ancient times, sea cucumbers have been regarded as excellent nutritional health foods, with the effects of enhancing muscle strength, enhancing immunity, anti-tumor, preventing arthritis and reducing blood lipid. Protein is an important component of sea cucumbers. In dried sea cucumbers, the protein content is close to 90%, which contains a lot of collagen. Sea cucumber peptides refer to small molecule peptides obtained by protease hydrolysis, separation and purification. Sea cucumbers also contain various active peptides such as neuropeptides, glycopeptides and antibacterial peptides. Sea cucumber peptides have various effects and play an irreplaceable role in human physiological functions compared with other nutrients. Also, due to the special physical and chemical properties of sea cucumber peptides such as good solubility, stability, low viscosity, easy digestion and absorption, non-antigenicity, and food safety, sea cucumber peptides have a higher biological potency than other ordinary sea cucumber products.

[0003] The fishy smell of sea cucumber peptides is an important factor affecting the product acceptance. The methods for removing the fishy smell mainly include physical methods such as adsorption, masking and embedding. However, the adsorption method will cause a certain loss of the original flavor of sea cucumbers, and the masking and embedding methods will result in a relatively low proportion of sea cucumber components, and there is a phenomenon of fishy smell return after the product is stored for a period of time.

[0004] The body wall of sea cucumbers is mostly brown or dark brown, with a large amount of pigments. The color of its protease hydrolysate is dark gray, which not only affects the appearance of the product, seriously affects the product quality, but also hinders the in-depth development of the product. Therefore, it is important to perform decolorization treatment on the sea cucumber polypeptide hydrolysate, which is beneficial to the in-depth development of the product. Currently, decolorizing agents such as powdered activated carbon, granular activated carbon, diatomite, macroporous adsorption resin, composite adsorbent, weakly basic anion resin, strongly basic anion resin, weakly acidic cation resin, etc. are often used to decolorize sea cucumber polypeptides. However, the traditional decolorization process is complex, with a high loss rate, and it will destroy the original structure and activity of sea cucumber peptides. Summary of the Invention

[0005] The present invention provides a preparation method of a probiotic sea cucumber peptide powder helpful for improving immunity. The probiotic sea cucumber peptide powder obtained by this method has no fishy smell, is light yellow in color and has a smooth luster, has a high number of viable probiotic bacteria, and at the same time maximally maintains the activity of the sea cucumber peptide powder, and there is no need for enzyme inactivation and decolorization treatment during the whole preparation process, such as adding adsorbents, activated carbon, etc.

[0006] The present invention provides a preparation method of a probiotic sea cucumber peptide powder helpful for improving immunity, which at least includes the following steps:

[0007] (1) In the homogenate of sea cucumber body wall, first add neutral protease for enzymatic hydrolysis, and then add papain for enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0008] (2) Add probiotics to the enzymatic hydrolysate for fermentation, and take the supernatant obtained from the fermentation;

[0009] Among them, the probiotics in step (2) are Eurotium cristatum with the preservation number of CICC No. 2099 and / or Bacillus coagulans with the preservation number of CGMCC No. 21801.

[0010] Further, the homogenate of sea cucumber body wall refers to the slurry obtained by cutting the sea cucumber body wall into pieces and then homogenizing with water. In the present invention, the homogenate of sea cucumber body wall can be obtained by washing dried sea cucumbers, cutting them into small pieces, adding water for pulping, homogenizing, and filtering.

[0011] Specifically, first add neutral protease to the homogenate of sea cucumber body wall for the first enzymatic hydrolysis. The enzymatic hydrolysis time is 1 - 2 hours, the enzymatic hydrolysis temperature is 50 - 55 °C, and the enzymatic hydrolysis pH is 6.5 - 7.5; then add papain for the second enzymatic hydrolysis. The enzymatic hydrolysis time is 2 - 3 hours, the enzymatic hydrolysis temperature is 50 - 55 °C, and the enzymatic hydrolysis pH is 6.5 - 7.5; the total enzymatic hydrolysis time is 3 - 5 h.

[0012] Further, the dosage ratio of neutral protease to papain in the above step (1) is 1 - 5:1 based on the enzyme activity.

[0013] Preferably, the dosage ratio of neutral protease to papain in the above step (1) is 1:1 based on the enzyme activity.

[0014] Further, the total addition amount of neutral protease and papain in the above step (1) is 6000 - 9000 U / g, the enzymatic hydrolysis temperature is 50 - 55 °C, the total enzymatic hydrolysis time is 3 - 5 h, and the enzymatic hydrolysis pH is 6.5 - 7.5.

[0015] Specifically, in step (2), the probiotics are inoculated into a fermenter for fermentation, and the probiotics are Eurotium cristatum (preservation number CICC No.2099 ), or Bacillus coagulans (preservation number CGMCC No.21801 ), or a mixture of Eurotium cristatum and Bacillus coagulans.

[0016] Further, the addition amount of Eurotium cristatum in step (2) is 0.02 - 0.04% of the mass of the enzymatic hydrolysate.

[0017] Further, the addition amount of Bacillus coagulans in step (2) is 0.02 - 0.04% of the mass of the enzymatic hydrolysate.

[0018] Further, the fermentation temperature in the above step (2) is 36 - 38 °C, and the fermentation time is 40 - 60 min.

[0019] Further, the probiotics in the above step (2) are a mixture of Eurotium cristatum and Bacillus coagulans. The inoculation ratio of Eurotium cristatum and Bacillus coagulans is 1:1 - 3 in terms of the number of viable bacteria per unit volume (CFU, Colony - Forming Units), and the total addition amount is 0.02 - 0.04% of the mass of the enzymolysis solution.

[0020] Further, the probiotics in the above step (2) are a mixture of Eurotium cristatum and Bacillus coagulans. The Eurotium cristatum and Bacillus coagulans can be mixed first and then added to the enzymolysis solution for fermentation; or the Eurotium cristatum and Bacillus coagulans can be added to the enzymolysis solution for fermentation in sequence.

[0021] Further, the preparation method of the probiotic sea cucumber peptide powder further includes: collecting the supernatant obtained by fermentation in step (2) and performing concentrated low - temperature spray drying to obtain a high - quality probiotic sea cucumber peptide powder product.

[0022] In addition, the present invention also provides a probiotic sea cucumber peptide powder prepared by any of the above methods.

[0023] The technical solution of the present invention has the following advantages:

[0024] 1. Deodorization effect: After the sea cucumber enzymolysis solution is fully enzymolyzed and co - fermented by Eurotium cristatum and Bacillus coagulans, the relative content of 9 representative fishy substances in the sea cucumber peptide powder is effectively reduced from 49.97% to 2.78%.

[0025] 2. Decolorization effect: After the sea cucumber enzymolysis solution is co - fermented by Eurotium cristatum and Bacillus coagulans, the color changes from the original brown to a light yellow transparent liquid. The sea cucumber peptide powder obtained by concentrating the supernatant after fermentation and performing low - temperature spray drying is milky yellow and has a smooth color. The decolorization effect is shown in Figure 2 . This solution omits the decolorization process flow, is simple to operate, and reduces costs.

[0026] 3. Peptide powder activity: The sea cucumber body wall homogenate is first enzymolyzed by neutral protease and papain. After enzymolysis, there is no need to perform high - temperature enzyme inactivation. Instead, Eurotium cristatum and Bacillus coagulans directly use protease as a nutrient for co - fermentation. After fermentation, there is no need to perform sterilization, and directly perform low - temperature spray drying to retain viable probiotic bacteria. The number of viable probiotic bacteria is greater than 1×10 9 per gram. And the entire process flow is completed at low temperature, maximizing the activity of the sea cucumber peptide powder and significantly improving the body's immunity.

[0027] 4. Flavor sensory: The enzymolysis solution of sea cucumber is co-fermented by Eurotium cristatum and Bacillus coagulans, which not only has a good deodorizing effect, but also produces a pleasant fermentation aroma and mellow fragrance, with a high sensory evaluation. Description of the Drawings

[0028] Figure 1 Effects of fermentation with different strains on the sensory score and fishy smell of sea cucumber peptide enzymolysis solution: a represents the sensory score and fishy smell of the enzymolysis solution of sea cucumber peptide fermented by Lactobacillus acidophilus, b represents the sensory score and fishy smell of the enzymolysis solution of sea cucumber peptide fermented by Eurotium cristatum, c represents the sensory score and fishy smell of the enzymolysis solution of sea cucumber peptide fermented by Bifidobacterium longum, d represents the sensory score and fishy smell of the enzymolysis solution of sea cucumber peptide fermented by Lactobacillus casei, e represents the sensory score and fishy smell of the enzymolysis solution of sea cucumber peptide fermented by Lactobacillus gasseri, f represents the sensory score and fishy smell of the enzymolysis solution of sea cucumber peptide fermented by Bacillus coagulans, g represents the sensory score and fishy smell of the enzymolysis solution of sea cucumber peptide co-fermented by Eurotium cristatum and Bacillus coagulans.

[0029] Figure 2 Photos of deodorized sea cucumber peptide powder: a represents the photo of deodorized sea cucumber peptide powder by Eurotium cristatum and Bacillus coagulans, b represents the photo of deodorized sea cucumber peptide powder by Eurotium cristatum, c represents the photo of deodorized sea cucumber peptide powder by Bacillus coagulans, d represents the photo of unfermented sea cucumber peptide powder.

[0030] Figure 3 High performance liquid chromatography (HPLC) chromatogram of the deodorized sea cucumber peptide powder described in the embodiment of the present invention.

[0031] Figure 4 Protein recovery rate and degree of hydrolysis of sea cucumber peptide hydrolyzed by adding neutral protease first and then papain.

[0032] Figure 5 Protein recovery rate and degree of hydrolysis of sea cucumber peptide hydrolyzed by adding papain first and then neutral protease.

[0033] Figure 6 Protein recovery rate and degree of hydrolysis of sea cucumber peptide hydrolyzed by neutral protease and papain simultaneously. Detailed Description of the Invention

[0034] The following embodiments are provided to better further understand the present invention, which are not limited to the best embodiment, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0035] Example 1 Preparation of sea cucumber peptide powder by co-fermentation of Eurotium cristatum and Bacillus coagulans

[0036] (1) Pretreatment: Wash the dried sea cucumbers, soak them at room temperature for 24 hours, change the water every 4 hours, and then clean the inner and outer surfaces of the sea cucumber body wall with clean water.

[0037] (2) Beating: Drain the sea cucumber body wall treated in step (1), cut into small pieces of (1~2) cm × (1~2) cm, add water to beat, homogenize, and filter. The filtrate obtained is the sea cucumber body wall homogenate, which is set aside.

[0038] (3) Enzymolysis: The filtrate prepared in step (2) was hydrolyzed by adding a composite enzyme: neutral protease was first added for the first enzymolysis, the enzymolysis time was 1 hour, the enzymolysis temperature was 50°C, and the enzymolysis pH was 7; then papain was added for the second enzymolysis, the enzymolysis time was 2.5 hours, the enzymolysis temperature was 50°C, and the enzymolysis pH was 7. The total amount of the composite enzyme added was 8000 U / g, and the composite enzyme ratio based on enzyme activity was: papain: neutral protease = 1:1.

[0039] (4) Microbial fermentation: The enzymatic hydrolysate obtained in step (3) was transferred to a fermentation tank, and direct-inoculation fermentation bacteria species Eurotium cristatum and Bacillus coagulans were added. The inoculation ratio was 1:1.5 based on the number of viable bacteria per unit volume. The total amount of probiotics added was 0.03% of the mass of the fermented liquid. Fermentation was carried out at 37°C for 50 min.

[0040] (5) The fermentation liquid after treatment in step (4) is concentrated and then spray-dried at low temperature to obtain a probiotic deodorized sea cucumber peptide powder rich in Eurotium cristatum and Bacillus coagulans.

[0041] Example 2 Preparation of Sea Cucumber Peptide Powder by Fermentation of Eurotium cristatum

[0042] (1) Pretreatment: Wash the dried sea cucumbers, soak them at room temperature for 24 hours, change the water every 4 hours, and then clean the inner and outer surfaces of the sea cucumber body wall with clean water.

[0043] (2) Beating: Drain the sea cucumber body wall treated in step (1), cut it into small pieces of (1-2) cm × (1-2) cm, add water, beat it, homogenize it, and filter it. The filtrate obtained is the sea cucumber body wall homogenate, which is set aside.

[0044] (3) Enzymolysis: The filtrate prepared in step (2) was hydrolyzed by adding a composite enzyme: neutral protease was first added for the first enzymolysis, the enzymolysis time was 1 hour, the enzymolysis temperature was 50°C, and the enzymolysis pH was 7; then papain was added for the second enzymolysis, the enzymolysis time was 1.5 hours, the enzymolysis temperature was 50°C, and the enzymolysis pH was 7. The total amount of the composite enzyme added was 8000 U / g, and the ratio of the composite enzyme activity of papain:neutral protease = 1:1.

[0045] (4)Microbial fermentation: Transfer the enzymolysis solution obtained in step (3) to a fermentation tank, and add the direct-inoculum fermentation strain Eurotium cristatum, with the addition amount being 0.03% of the mass of the liquid to be fermented. Ferment at 37 °C in the fermentation tank for 50 min.

[0046] (5)Perform low-temperature spray drying on the fermented and concentrated liquid processed in step (4) to obtain the finished product of probiotic deodorized sea cucumber peptide powder rich in Eurotium cristatum.

[0047] Example 3 Preparation of sea cucumber peptide powder by Bacillus coagulans fermentation

[0048] (1)Pretreatment: Wash the dried sea cucumbers, soak them at room temperature for 24 h, change the water every 4 h, and then wash the inner and outer surfaces of the sea cucumber body wall with clean water.

[0049] (2)Pulping: Drain the sea cucumber body wall processed in step (1), cut it into small pieces of (1-2) cm × (1-2) cm, add water for pulping, homogenize, and filter. The obtained filtrate is the homogenate of the sea cucumber body wall for later use.

[0050] (3)Enzymolysis: Add a composite enzyme to the filtrate prepared in step (2) for stepwise enzymolysis: First, add neutral protease for the first enzymolysis, with the enzymolysis time being 1 hour, the enzymolysis temperature being 50 °C, and the enzymolysis pH being 7; then add papain for the second enzymolysis, with the enzymolysis time being 1.5 hours, the enzymolysis temperature being 50 °C, and the enzymolysis pH being 7. The total addition amount of the composite enzyme is 8000 U / g, and the compound enzyme activity ratio of the composite enzyme is papain:neutral protease = 1:1.

[0051] (4)Microbial fermentation: Transfer the enzymolysis solution obtained in step (3) to a fermentation tank, add the direct-inoculum fermentation strain Bacillus coagulans, with the addition amount being 0.03% of the mass of the liquid to be fermented. Ferment at 37 °C in the fermentation tank for 50 min.

[0052] (5)Perform low-temperature spray drying on the concentrated liquid processed in step (4) to obtain the finished product of probiotic deodorized sea cucumber peptide powder rich in Bacillus coagulans.

[0053] Example 4 Preparation of unfermented sea cucumber peptide powder

[0054] (1)Pretreatment: Wash the dried sea cucumbers, soak them at room temperature for 24 h, change the water every 4 h, and then wash the inner and outer surfaces of the sea cucumber body wall with clean water.

[0055] (2)Pulping: Drain the sea cucumber body wall processed in step (1), cut it into small pieces of (1-2) cm × (1-2) cm, add water for pulping, homogenize, and filter. The obtained filtrate is the homogenate of the sea cucumber body wall for later use.

[0056] (3) Enzymatic hydrolysis: Place the filtrate prepared in step (2) in a beaker and add a composite enzyme for stepwise enzymatic hydrolysis: First, add neutral protease for the first enzymatic hydrolysis. The enzymatic hydrolysis time is 1 hour, the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis pH is 7. Then add papain for the second enzymatic hydrolysis. The enzymatic hydrolysis time is 1.5 hours, the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis pH is 7. The total addition amount of the composite enzyme is 8000 U / g, and the ratio of the composite enzyme's complex enzyme activity is papain:neutral protease = 1:1.

[0057] (4) Enzyme inactivation: Inactivate the enzyme solution processed in step (3) by heating it in a boiling water bath at 90 °C for 15 min, and then cool it to room temperature.

[0058] (5) Centrifugation: Centrifuge the enzyme solution processed in step (4) and retain the supernatant. The centrifugation conditions are: 3500 rpm / min for 20 min.

[0059] (6) Concentration: Concentrate the supernatant collected in step (5) to 5 - 10% of the original volume.

[0060] (7) Drying: Subject the concentrated solution processed in step (6) to low-temperature spray drying to obtain the finished product of sea cucumber peptide powder.

[0061] Example 5 Preparation of sea cucumber peptide powder by fermentation of Lactobacillus acidophilus

[0062] The same as Example 2, except that the probiotic is Lactobacillus acidophilus

[0063] Example 6 Preparation of sea cucumber peptide powder by fermentation of Bifidobacterium longum

[0064] The same as Example 2, except that the probiotic is Bifidobacterium longum

[0065] Example 7 Preparation of sea cucumber peptide powder by fermentation of Lactobacillus casei

[0066] The same as Example 2, except that the probiotic is Lactobacillus casei

[0067] Example 8 Preparation of sea cucumber peptide powder by fermentation of Lactobacillus gasseri

[0068] The same as Example 2, except that the probiotic is Lactobacillus gasseri

[0069] Example 9 Detection and analysis of fishy odor substances in sea cucumber peptide powder prepared by different methods

[0070] For the four products in Example 1, Example 2, Example 3 and Example 4, the volatile flavor substances were determined by GC-MS as shown in Table 1, and the comparative analysis results of the relative percentage contents of the representative fishy odor substances are shown in Table 2.

[0071] Comparative Analysis Results of the Relative Percentage Contents of Volatile Components in the Products of Four Examples

[0072]

[0073] As can be seen from Table 1, a total of 107 aroma substances were detected. Among them, 65 were detected in the unfermented sea cucumber peptides of Example 4, and these compounds were mainly aldehydes and alkanes. In the Eurotium cristatum - deodorized sea cucumber peptide powder of Example 2, 41 aroma substances were detected, mainly alcohols and lipids. Compared with Example 4, the Eurotium cristatum - deodorized sea cucumber peptide had 5 more alcohols, and the relative content of alcohols in the Eurotium cristatum - deodorized sea cucumber peptide powder increased by 2.20 times compared with Example 4. In the Bacillus coagulans - deodorized sea cucumber peptide powder of Example 3, 50 aroma substances were detected. Finally, in the Eurotium cristatum and Bacillus coagulans - deodorized sea cucumber peptide of Example 1, 52 aroma substances were detected, mainly alcohols and lipids. It shows that the metabolic activities of the strains Eurotium cristatum and Bacillus coagulans both produce alcohol substances. The main aroma characteristics of alcohol substances are fruity and grassy aromas, and the increase in alcohol substances after fermentation makes the sea cucumber peptide have more pleasant aromas.

[0074] Comparative Analysis Results of the Relative Percentage Contents of Representative Odorous Substances in Four Sea Cucumber Peptide Powders

[0075]

[0076] As can be seen from Table 2, the representative odorous substances in the unfermented sea cucumber peptide powder of Example 4 are (E)-2, (Z)-6 - nonadienal, (E)-2 - octenal, decanal, nonanal, n - octanal, 1 - octen - 3 - ol, n - hexanal, heptanal, and n - heptanol, which make the sea cucumber peptide have fishy, earthy, and rancid odors. In the Eurotium cristatum - fermented sea cucumber peptide powder of Example 2, compared with the unfermented sea cucumber peptide powder of Example 1, decanal was not detected after fermentation. And the Eurotium cristatum - fermented and deodorized sea cucumber peptide powder of Example 2 reduced the relative content of the nine representative odorous substances in the unfermented sea cucumber peptide powder of Example 4 from 49.97% to 17.21%. In the Bacillus coagulans - fermented and deodorized sea cucumber peptide of Example 3, compared with the unfermented sea cucumber peptide of Example 4, the relative content of the nine representative odorous substances decreased from 49.97% to 27.92%. In the Eurotium cristatum and Bacillus coagulans - compound - deodorized sea cucumber peptide powder of Example 1, compared with the unfermented sea cucumber peptide of Example 4, decanal, (E)-2 - octenal, and decanal were not detected after fermentation, and the relative content of the nine representative odorous substances decreased from 49.97% to 2.78%.

[0077] Referring to the aforementioned detection method, the results of the fishy smell detection of the sea cucumber peptide powder described in Examples 1-7 are as follows Figure 1 shown.

[0078] Comparative analysis of the decolorization of sea cucumber peptide powder prepared by different methods in Example 10

[0079] The color of the sample was measured at room temperature using a WSC-S colorimeter. L*, a*, and b* represent color coordinates, and the Hunter whiteness calculation method was used. L* represents the brightness of the color, and a*, b* represent the color direction. The parameters of the standard whiteboard used were: L* = 93.61, a* = 0.89, b* = 1.43. The color results of Examples 1-4 are shown in Table 3.

[0080] Table 3 Color of sea cucumber peptide powder in four examples

[0081]

[0082] Note: Different letters in each column represent significant differences between two groups of data.

[0083] As can be seen from Table 3, the L*, a*, and b* values of the sea cucumber peptide powder obtained in different examples are different, indicating that the preparation method of the sea cucumber peptide powder in the examples has a greater impact on the color. Among them, there is a significant difference between Example 1 and Example 4. Compared with Example 4, the Eurotium cristatum and Bacillus coagulans-deodorized sea cucumber peptide powder in Example 1 is milky yellow and has a smooth color.

[0084] Comparative analysis of the effects of different methods of preparing sea cucumber peptide powder on immunity in Example 11

[0085] An immune-suppressed mouse model was established by cyclophosphamide induction to evaluate whether four sea cucumber peptide powder products, namely Example 1, Example 2, Example 3, and Example 4, can help improve the immunity of immune-suppressed mice. The specific animal experiment is as follows:

[0086] The clean-grade Kunming mice were divided into 6 groups, namely the blank group, the model group, the Eurotium cristatum and Bacillus coagulans-deodorized sea cucumber peptide powder group in Example 1, the Eurotium cristatum-deodorized sea cucumber peptide powder in Example 2, the Bacillus coagulans-deodorized sea cucumber peptide powder in Example 3, and the unfermented sea cucumber peptide powder group in Example 4, with 12 mice in each group. The gavage dose of the sea cucumber peptide powder was 5 mg·kg -1 . Each group was gavaged once a day. The blank group and the model group were gavaged with an equal amount of normal saline for 14 consecutive days. On the 8th, 9th, and 10th days of the experiment, the model group and the sea cucumber peptide powder group were intraperitoneally injected with 80 mg·kg -1 cyclophosphamide solution for three consecutive days to establish an immune-suppressed model. The mice were fasted and dehydrated for 12 h after the last administration, weighed, sacrificed by cervical dislocation, the chest and abdomen were opened, and the spleen and thymus were taken and weighed. The spleen index and thymus index were calculated. The calculation formulas are as follows:

[0087]

[0088] The spleen and thymus are important immune organs of the human body, and the changes in thymus and spleen indices are important indicators of body immunity. The results of the effects of sea cucumber peptide powder in each example group on the body weight and organ indices of immunosuppressed mice are shown in Table 4.

[0089] Table 4 Effects of sea cucumber peptide powder obtained from examples on the body weight and organ indices of immunosuppressed mice

[0090]

[0091] Note: Data are expressed as mean ± SD, n = 12, and different letters in each column represent significant differences between two groups of data.

[0092] It can be seen from the results in Table 4 that there was no significant difference in the initial body weights of mice among groups. During the experiment, the final body weight of the model group decreased extremely significantly compared with that of the normal group. The final body weights of the mice in the intragastric administration groups of Example 1 and Example 2 showed an increasing trend compared with the model group, and the increase in the Example 1 group was extremely significant. It can be known from the results that Example 2 can significantly promote the weight gain of immunosuppressed mice; among them, Example 1 can extremely significantly promote the weight gain of immunodeficient mice.

[0093] It can be seen from Table 4 the effects of sea cucumber peptide powder obtained from examples on the spleen and thymus indices of immunodeficient mice. Compared with the normal group, the spleen index and thymus index of the model group were extremely significantly lower than those of the normal group, indicating that the injection of cyclophosphamide led to the atrophy of immune organs. After intragastric administration of sea cucumber peptide powder of Examples 1-4 to immunodeficient mice, compared with the model group, the spleen index and thymus index of the mice in the Example 1-2 groups showed an increasing trend, and the spleen index of the mice in the Example 1 group increased extremely significantly. It can be speculated that the sea cucumber peptide powder obtained from Example 1 can effectively relieve the atrophy and damage of the organ indices of immunodeficient mice caused by cyclophosphamide, and has a significant effect on improving immunity.

[0094] Comparative analysis of the sensory evaluation of sea cucumber peptide powder prepared by different methods in Example 12

[0095] For the four products in Example 1, Example 2, Example 3 and Example 4, after adding water and dissolving them, 12 people were invited for sensory evaluation. The sensory scoring form is shown in Table 5. The sensory score of a compound bacteria-deodorized sea cucumber peptide powder of Eurotium cristatum and Bacillus coagulans was the highest, 93.6 points; the sensory score of a deodorized sea cucumber peptide powder of Eurotium cristatum was the second, 75.5 points; next was a deodorized sea cucumber peptide powder of Bacillus coagulans, with a sensory score of 73.5 points; the lowest sensory score was for an unfermented sea cucumber peptide powder, with a sensory score of 66.3 points; the specific data of the deodorized sea cucumber peptides are shown in Table 6. Referring to the foregoing evaluation method, the sensory evaluation results of the sea cucumber peptide powders described in Examples 1-7 are as Figure 1 shown.

[0096] Table 5 Sensory Scoring Form

[0097]

[0098] Table 6 Sensory Evaluation Results of Four Kinds of Sea Cucumber Peptide Powders

[0099] Effect of Sea Cucumber Peptide Powders Prepared by Different Enzymatic Hydrolysis Processes in Example 13 on Protein Recovery Rate and Degree of Hydrolysis

[0100] 1. Preparation of Sea Cucumber Peptide Powder by Stepwise Enzymatic Hydrolysis (First Add Neutral Protease and Then Papain)

[0101] (1) Pretreatment: Wash the dried sea cucumbers, soak them at room temperature for 24 h, change the water every 4 h, and then wash the inner and outer surfaces of the sea cucumber body wall with clean water.

[0102] (2) Pulping: Drain the sea cucumber body wall processed in step (1), cut it into small pieces of (1-2) cm×(1-2) cm, add water for pulping, homogenize, and filter. The obtained filtrate is the homogenate of the sea cucumber body wall for later use.

[0103] (3) Enzymatic hydrolysis: Place the filtrate prepared in step (2) in a beaker and add a compound enzyme for stepwise enzymatic hydrolysis: First add neutral protease for the first enzymatic hydrolysis, with an enzymatic hydrolysis time of 1 hour, an enzymatic hydrolysis temperature of 50 °C, and an enzymatic hydrolysis pH of 7; then add papain for the second enzymatic hydrolysis, with an enzymatic hydrolysis time of 1.5 hours, an enzymatic hydrolysis temperature of 50 °C, and an enzymatic hydrolysis pH of 7. The total addition amount of the compound enzyme is 8000 U / g, and the compound enzyme activity ratio of papain:neutral protease = 1:1. Take the supernatant to obtain the enzymatic hydrolysate.

[0104] 2. Preparation of Deodorized Sea Cucumber Peptides by Stepwise Enzymatic Hydrolysis (First Add Papain and Then Neutral Protease)

[0105] (1) Pretreatment: Wash the dried sea cucumbers, soak them at room temperature for 24 h, change the water every 4 h, and then wash the inner and outer surfaces of the sea cucumber body wall with clean water.

[0106] (2) Pulping: Drain the sea cucumber body wall processed in step (1), cut it into small pieces of (1 - 2) cm × (1 - 2) cm, add water for pulping, homogenize, and filter.

[0107] (3) Enzymatic hydrolysis: Place the ground slurry prepared in step (2) in a beaker, and add a composite enzyme for stepwise enzymatic hydrolysis: First, add papain for the first enzymatic hydrolysis, with an enzymatic hydrolysis time of 1.5 hours, an enzymatic hydrolysis temperature of 50 °C, and an enzymatic hydrolysis pH of 7; then add neutral protease for the second enzymatic hydrolysis, with an enzymatic hydrolysis time of 1 hour, an enzymatic hydrolysis temperature of 50 °C, and an enzymatic hydrolysis pH of 7. The total addition amount of the composite enzyme is 8000 U / g, and the compound enzyme activity ratio of the composite enzyme is papain:neutral protease = 1:1. Take the supernatant to obtain the enzymatic hydrolysate.

[0108] 3. Preparation of deodorized sea cucumber peptides by synergistic enzymatic hydrolysis (adding neutral protease and papain simultaneously)

[0109] (1) Pretreatment: Wash the dried sea cucumbers, soak them at room temperature for 24 hours, change the water every 4 hours, and then wash the inner and outer surfaces of the sea cucumber body wall with clean water.

[0110] (2) Pulping: Drain the sea cucumber body wall processed in step (1), cut it into small pieces of (1 - 2) cm × (1 - 2) cm, add water for pulping, homogenize, and filter.

[0111] (3) Enzymatic hydrolysis: Place the ground slurry prepared in step (2) in a beaker, and simultaneously add papain and neutral protease for synergistic enzymatic hydrolysis: The enzymatic hydrolysis time is 3 hours, the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis pH is 7. The total addition amount of the composite enzyme is 8000 U / g, and the compound enzyme activity ratio of the composite enzyme is papain:neutral protease = 1:1. Take the supernatant to obtain the enzymatic hydrolysate.

[0112] For the above three enzymatic hydrolysates, the degree of hydrolysis was determined by the o - phthalaldehyde (OPA) method, and the protein recovery rate was determined by the Kjeldahl method. The specific data are shown in Figures 4 - 6 . It can be seen from Figure 4 that when the ratio of papain to neutral protease is 1:1.5 and 1:1, the protein recovery rate and degree of hydrolysis of sea cucumber protein hydrolyzed by adding neutral protease first and then papain are 14.8% and 79%, 12.9% and 97% respectively; it can be obtained from Figure 5 that when the ratio of papain to neutral protease is 1:1, the degree of hydrolysis and recovery rate of sea cucumber protein hydrolyzed by adding papain first and then neutral protease are 14.3% and 76%; Figure 6It shows that when the ratio of papain to neutral protease is 1:1, the hydrolysis degree and recovery rate of sea cucumber protein hydrolyzed by adding neutral protease and papain simultaneously are 14.6% and 81%. Considering the protein recovery rate and hydrolysis degree comprehensively, the condition of adding neutral protease first and then papain with the enzyme ratio (papain:neutral protease) of 1:1 is the optimal one.

Claims

1. A preparation method of probiotic sea cucumber peptide powder that helps to improve immunity, characterized in that, It includes the following steps: (1) In the homogenate of sea cucumber body wall, first add neutral protease for enzymatic hydrolysis, and then add papain for enzymatic hydrolysis to obtain an enzymatic hydrolysate; (2) Add probiotics to the enzymatic hydrolysate for fermentation, and take the supernatant obtained by fermentation; (3) Collect the supernatant obtained by fermentation in step (2), concentrate it, and perform low-temperature spray drying to obtain probiotic sea cucumber peptide powder; Among them, in step (1), the ratio of the neutral protease to the papain is 1-5:1 in terms of enzyme activity, the total addition amount of the neutral protease and the papain is 6000-9000 U / g, the enzymatic hydrolysis temperature is 50-55°C, the enzymatic hydrolysis time is 3-5 h, and the enzymatic hydrolysis pH is 6.5-7.5; The probiotic bacteria described in step (2) are Eurotium cristatum with the preservation number of CICC No.2099 and Bacillus coagulans with the preservation numbers of CGMCC No.21801 ; In step (2), the fermentation temperature of the fermentation is 36-38°C, the fermentation time is 40-60 min, and for Eurotium cristatum and Bacillus coagulans, calculated by the number of viable bacteria per unit volume, the inoculation ratio is Eurotium cristatum: Bacillus coagulans = 1:1-3, and the total addition amount is 0.02-0.04% of the mass of the enzymatic hydrolysate.

2. The preparation method of the probiotic sea cucumber peptide powder for enhancing immunity according to claim 1, characterized in that, In step (2), the addition process of Eurotium cristatum and Bacillus coagulans is to mix Eurotium cristatum and Bacillus coagulans first and then add them to the enzymatic hydrolysate in step (1) for fermentation; or add Eurotium cristatum and Bacillus coagulans to the enzymatic hydrolysate in step (1) for fermentation separately in sequence.

3. A probiotic sea cucumber peptide powder that helps improve immunity, characterized in that, Prepared by the preparation method according to any one of claims 1-2.

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