CN112544874A

CN112544874A - Nutritional noodles added with fish polypeptide powder and preparation method thereof

Info

Publication number: CN112544874A
Application number: CN202011303922.1A
Authority: CN
Inventors: 朴美子; 李震; 张月
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2021-03-26

Abstract

The invention discloses a nutritional noodle added with fish polypeptide powder and a preparation method thereof. The nutritional noodles comprise fish polypeptide powder and flour which are obtained by fermenting fish leftovers with Bacillus beijerinckii MRS with the preservation number of CGMCC No.18065 and then performing enzymolysis with alkaline protease. The preparation method of the nutritional noodles is simple, peptides in the fish directly enter cells in a small molecular peptide form to promote nutrient absorption, and the fish polypeptide powder is added into the noodles, so that the taste of the noodles is enriched, the noodles are endowed with higher nutritional value, the fish leftovers are fully utilized, the waste of materials is avoided, the cohesiveness, the elasticity and the toughness of the noodles are enhanced, and the viscoelasticity and the strength of the noodles are reduced.

Description

Nutritional noodles added with fish polypeptide powder and preparation method thereof

Technical Field

The invention relates to the field of food, in particular to nutritional noodles added with fish polypeptide powder and a preparation method thereof.

Background

The noodles are taken as a traditional food which has a history of more than four thousand years, is simple to prepare and is convenient to eat, and are deeply accepted and loved by people. However, the ingredients and the taste of the noodles on the market are single at present, and although certain energy can be supplemented after the noodles are eaten, the nutritional value is not high.

The fish meat is rich in animal protein, phosphorus and the like, is rich in nutrition, delicious in taste, easy to digest and absorb by human bodies, and has an important effect on the development of human physical strength and intelligence, so that the fish meat is deeply popular with people; the fish bones, fish bones and the like which are left after eating the fish meat also contain rich high-quality protein and calcium, and can prevent and treat osteoporosis to a certain extent; the fish skin also contains abundant protein and multiple microelements, can supplement leucocyte for human body, and has certain anticancer effect. Wherein the cod is a cold water fish living in the bottom layer of the sea and the middle and lower layers of the deep sea, and has the advantages of high protein, low fat, delicious meat quality and the like. Cod contains rich nutrients such as: protein, vitamin A, vitamin D, calcium, magnesium, selenium and the like, and the cod also has certain health-care effects: the fish can promote blood circulation and remove blood stasis; swim bladder can nourish blood and stop bleeding; the fishbone can be used for treating loempe; the cod liver oil has effects of healing sore, clearing heat, diminishing inflammation, and inhibiting mycobacterium tuberculosis.

At present, fish meat is mostly directly processed into surimi, fish meal or fish paste and directly mixed into other materials to prepare fish additive food, but many nutrient substances in fish cannot directly enter cells to play a role, so that the absorption and utilization of nutrition in fish are not facilitated, and most fishbone skin cannot be effectively utilized, so that the waste of resources is caused.

Disclosure of Invention

In order to overcome and solve the defects in the prior art, the invention provides nutritional noodles added with fish polypeptide powder and a preparation method thereof. The nutritional noodles are prepared from polypeptide powder and flour which are produced by fermenting and enzymolyzing fish leftovers, and have unique taste and higher nutritional value.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides nutritional noodles added with fish polypeptide powder, which comprise fish polypeptide powder and flour.

Furthermore, the fish polypeptide powder is obtained by fermenting fish leftovers by bacillus belief and then performing enzymolysis by alkaline protease.

Further, the Bacillus belgii adopts Bacillus belgii MRS with the preservation number of CGMCC No. 18065.

Furthermore, the thallus of the Bacillus belgii MRS is long rod-shaped and slender, the bacterial colony is round, faint yellow and semitransparent, has a regular shape, a convex surface, is moist and smooth and is in a mucus shape, and the diameter of the bacterial colony is 1.5-2.5 mm.

The invention also provides a preparation method of the nutritional noodles, which comprises the following steps:

(1) after activation of Bacillus beleisi, inoculating the activated Bacillus beleisi in MRS liquid culture medium at 28-30 ℃, and performing shaking culture at 150-180rpm/min for 24-48 h until the concentration is 6-8 × 10⁷cfu/mL to obtain Bacillus beiLeisi seed liquid;

(2) crushing and refining fish leftovers by using a crusher, adding distilled water according to a feed-liquid ratio (mass unit is g, volume unit is ml) of 1:1-3:1 for sterilization, cooling to room temperature to obtain fish leftover slurry, adding Bacillus beidellis seed liquid according to the addition amount of 3-4% of the mass of the fish leftover slurry, fermenting at 28-30 ℃ and 180rpm/min for 24-36h to obtain a fish fermentation product;

(3) adding alkaline protease into the fish fermentation product in an adding amount of 200-;

(4) mixing the fish polypeptide powder with flour in an amount of 3-5% of the flour mass, adding water 40-50% of the flour mass into a noodle maker, kneading for 15-20min at 25-30 deg.C, keeping humidity, standing at 25-30 deg.C for 20-30min, pressing into noodles by a noodle press, dehydrating, and drying to obtain the nutritional noodles.

Further, the fish leftovers comprise at least one of fish skin, fish meat and fish bones.

Further, the fish polypeptide powder comprises at least one of fish skin polypeptide powder, fish meat polypeptide powder and fish bone polypeptide powder.

Further, the fish includes cod, salmon, barberry, and tuna.

Preferably, the fish is cod.

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

1. the cod leftovers are fermented by using Bacillus belgii MRS, then are subjected to enzymolysis by using alkaline protease, are concentrated and are freeze-dried to obtain cod polypeptide powder, and the cod polypeptide powder is mixed with flour and water to prepare the nutritional noodles. The cod peptide in the nutritional noodles can directly enter cells in the form of small molecular peptide, the enzyme activity can be excited, the nutrition can be supplemented, and the cod polypeptide powder is added into the noodles, so that the taste of the noodles is enriched, the noodles are endowed with higher nutritional value, the noodles are more beneficial to the absorption of nutrition of a human body, cod leftovers are fully utilized, and the waste of materials is avoided.

2. The nutritious noodles added with the cod polypeptide powder are verified by the invention, so that the cohesiveness, elasticity and toughness of the noodles are enhanced, the noodles are smooth and chewy, and the flavor and nutrition of the noodles are increased; the viscoelasticity and the strength of the noodles are reduced, and the noodles are more suitable for children and old people; the nutritional noodles have unique taste, rich nutrition and safe eating.

Drawings

FIG. 1: photographs of colonies of Bacillus belgii MRS on plates.

FIG. 2: a 16SrDNA homology phylogenetic tree of bacillus belgii MRS.

FIG. 3: the influence of the inoculation amount on the content of polypeptide produced by fermenting the cod leftovers.

FIG. 4: the influence of the feed liquid on the content of polypeptide produced by fermenting the cod leftovers is reduced.

FIG. 5: the influence of the fermentation time on the content of polypeptide produced by fermenting the cod leftovers.

FIG. 6: influence of the amount of alkaline protease on the content of polypeptide in the enzymatic hydrolysate.

FIG. 7: influence of enzymolysis time on the polypeptide content in the enzymolysis product.

FIG. 8: and (4) measuring the antioxidant activity under the optimal fermentation and enzymolysis conditions.

FIG. 9: influence of different addition amounts of fishbone polypeptide powder on dough gelatinization characteristics.

FIG. 10: the elastic modulus G 'and viscous modulus G' of the mixed dough at different addition levels are plotted against the frequency Hz.

FIG. 11: the elastic modulus G 'and viscous modulus G' of the dough were mixed at different addition levels as a function of temperature.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1: screening and identification of Bacillus beleisi MRS

1. Primary screen for bacteria

After smashing Pu' er tea cake produced in 2005, Meng Hai, Yunnan, 2g of tea leaves were weighed and poured into a conical flask containing 18mL of sterile water for thorough mixing. Sucking 1mL of tea stock solution, adding into a test tube containing 9mL of sterile water, and diluting to 10%^-2、10^-3And 10^-4Gradient of (2), removing 10^-2、10^-3And 10^-40.1mL of each of the solutions was plated on a previously prepared LB solid medium plate, finally plated (2 plates per concentration), and cultured in an incubator at 37 ℃ for 24 hours, to compare the growth of colonies on the plate.

2. Bacterial rescreening

Selecting single colonies with different growth forms from an LB medium plate to draw lines in four areas in an aseptic environment, repeatedly drawing the lines for many times, performing gram staining on bacteria, checking the form of the bacteria under a 100-time oil scope until the bacteria are purified, inoculating the purified bacteria to an inclined plane, culturing for 24-48 h in an incubator, sealing and marking the bacteria with MRS names after the bacteria grow well on the inclined plane, and refrigerating and storing at-4 ℃.

3. Morphological and physiological biochemical identification

Observing and recording the shape and characteristics of a single MRS colony on a purified culture medium plate, then performing gram staining, observing under a 100-fold oil microscope, recording the observed bacterial morphology, and preliminarily identifying the strain MRS according to Bergey's Manual of bacteria and ' Manual of bacteria System identification '.

The result shows that the strain MRS is a gram-positive bacterium, the colony morphology and the characteristics of the strain MRS are shown in figure 1, and the bacterium body is long rod-shaped and relatively slender; the colony color is faint yellow, round, semitransparent, has a regular shape, a convex surface, is smooth and moist, is sticky and has a diameter of 2 mm. The colony is subjected to a citrate utilization test, an M.R. test, a V.P. test, an acid production test, a glucose, fructose and sucrose utilization test and a catalase test, and the results are positive, so that MRS is preliminarily judged to be bacillus. The strain MRS is aerobic at a suitable growth temperature of 20-42 ℃ and a suitable growth pH of 6.0-9.0.

4. Strain 16S rDNA sequence analysis

The CTAB combination method is selected to extract the genome DNA of the bacteria, lysozyme is added to dissolve the cell wall of the bacteria, and protease K and detergent are used to crack the bacteria to obtain the genome DNA. PCR amplification was performed with 16S rDNA universal primers. The primer sequences used were:

27F：5’-AGTTTGATCMTGGCTCAG-3’；

1540R：5’-AGGAGGTGATCCAGCCGCA-3’；

7F：5’-CAGAGTTTGATCCTGGCT-3’；

1492R：5’-GGTTACCTTGTTACGACTT-3’。

the PCR reaction system is as follows: 10 XBuffer 5 uL, dNTP 20 uL, primer 27F 1 uL, primer 1492R 1 uL, Taq enzyme 5 uL, DNA template 1 uL, adding water to 50 uL.

The PCR program was set to: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s, and extension at 72 ℃ for 20s for 30 cycles; extending for 8min at 72 ℃; keeping the temperature at 4 ℃.

After electrophoresis of the test strips, TA ligation was performed using pEASY-T1 as a vector, the ratio of the target fragment to the vector being 8: 1. After transfer to e.coli 110 strain at 42 ℃, the plate was spread on LB plates with ampicillin, the blue-white spots of the plate were observed, and desired colonies were selected for positive clones identified by PCR. Extracting DNA according to the kit, then transferring the DNA to a biological engineering company Limited for sequencing, comparing the sequencing result on NCBI through BLAST to find out similar sequences, then adopting Clustal software for comparison, and selecting MEGA to establish a phylogenetic tree of bacteria.

As shown in FIG. 2, the 16S rDNA homology phylogenetic tree of the strain MRS shows that the MRS strain and the Bacillus velezensis have a close relationship, so that the strain MRS is judged to be the Bacillus velezensis.

The invention carries out strain preservation on the screened strain MRS, and the preservation unit of the Bacillus beiLeisi MRS is as follows: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: year 2019, month 07, day 03; the preservation number of the Bacillus velezensis is CGMCC No. 18065.

Example 2: preparation method of nutritional noodles added with cod leftover polypeptide powder

A preparation method of nutritional noodles added with cod leftover polypeptide powder specifically comprises the following steps:

1. strain activation and preparation of seed liquid

Inoculating Bacillus beilis MRS in glycerol storage tube, performing plate streaking activation on LB plate, performing inversion culture at 30 deg.C for 24 hr, selecting single colony on the plate to LB slant culture medium, standing for 24 hr, selecting strain on LB slant culture medium, inoculating into MRS liquid culture medium, performing shake culture at 30 deg.C and 180r/min for 24 ^ to ^48h to a concentration of about 8X 10⁷cfu/mL to obtain Bacillus belgii seed solution.

2. Raw material treatment

Removing impurities such as fish scales and internal organs from the cod leftovers, respectively crushing the fish skin, the fish bones and the fish meat into fine powder by using a crusher, and then putting the fine powder into a refrigerator for freezing for later use, wherein the crushed mixture is used as a subsequent fermentation substrate.

3. Preparation of fermentation products

Respectively filling the cod skin, the fish bones and the fish meat into a 250mL conical flask, and respectively adding the weight percentages of 3:1, adding distilled water, sealing, sterilizing in a water bath kettle at 78 ℃ for 30min, cooling to room temperature to obtain fish skin pulp, fish bone pulp and fish meat pulp respectively, adding Bacillus beijerinckii seed liquid according to the addition amount of 3% of the mass of the fish skin pulp, the fish bone pulp and the fish meat pulp respectively, fermenting in a shaking table at 30 ℃ and 180rpm/min respectively, wherein the fish skin and the fish meat are fermented for 24h, and the fish bones are fermented for 36h to obtain a fermentation product.

4. Preparation of enzymatic hydrolysate

Respectively carrying out enzymolysis on the fermentation products by using alkaline protease, wherein the addition amount of the alkaline protease in the fish skin fermentation product is 200U/g, the pH value is 10, the temperature is 55 ℃, and the enzymolysis is carried out for 3h at the rotating speed of 180r/min, so as to obtain fish skin enzymolysis liquid; adding alkaline protease into the fish fermented product at 400U/g, pH 10, 55 deg.C, and rotating speed of 180r/min for 5 hr to obtain fish enzymatic hydrolysate; adding alkaline protease into fishbone fermentation product at 400U/g, pH 10, 55 deg.C, and rotating at 180r/min for 3 hr to obtain fishbone enzymolysis liquid; and respectively centrifuging the enzymatic hydrolysate, taking supernatant, concentrating, and freeze-drying to obtain polypeptide powder.

The fish bone fermentation product can also be directly used for preparing the nutritional noodles without using alkaline protease for enzymolysis.

5. Preparation method of nutritious noodle

Mixing the polypeptide powder with flour by the addition amount of 3%, adding water with the volume fraction of 50%, kneading for 15-20min at 25-30 ℃ by a noodle maker, standing the kneaded dough at the ambient temperature of 20-30 ℃ for 30min under the condition of keeping humidity, pressing into noodles with the required width and length by a noodle press according to actual requirements, and dehydrating and drying to obtain the codfish polypeptide nutritional noodles.

The polypeptide powder can be any one of fish skin, fish bone and fish polypeptide powder, or can be mixed with the fish skin polypeptide powder, the fish bone polypeptide powder and the fish polypeptide powder in any proportion.

Example 3: process optimization for producing polypeptide by fermenting cod leftovers through Bacillus belgii MRS

Influence of inoculation amount, feed-liquid ratio and fermentation time on polypeptide production of cod leftovers

1. Influence of inoculation amount on content of polypeptide produced by fermenting cod leftovers

Taking 20g of pasteurized fish skin, setting the ratio of material to liquid to be 1:5, respectively setting the inoculum sizes to be 2%, 4%, 6% and 8%, carrying out shake culture at the temperature of 30 ℃ and the rotating speed of 180r/min, fermenting for 24h, centrifuging at 4000r/min after the fermentation is finished, and measuring the peptide content in the supernatant. The above experimental procedures were repeated for fish and fishbone, respectively.

The polypeptide content of the sample is determined by using a BCA method: firstly, a BSA standard curve is drawn, and BSA standard substances are prepared into solutions with mass concentrations of 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5g/L by using physiological saline and placed in a centrifuge tube. And fully and uniformly mixing the reagent A and the reagent B in the BCA kit according to the volume ratio of 50: 1 to obtain a BCA working solution. 20 mu L of solution is absorbed from BSA tubes with various concentrations, and is fully mixed with 200 mu L of BCA working solution, the mixture is placed in a water bath with the temperature of 37 ℃ and shaken for 60min, distilled water is used as a blank, the light absorption value is measured at 562nm, and a BSA standard curve is drawn. When the content of the polypeptide in the sample is measured, 100 mu L of diluted sample solution with corresponding times is added into a test tube, 1000 mu L of BCA working solution is added at the same time, the mixture is quickly mixed, the mixture is placed in a water bath at 37 ℃ for 60min, the light absorption value is measured at 562nm, the test is repeated for 2 times, and the percentage content of the polypeptide in the sample is calculated according to a standard curve.

As a result, as shown in FIG. 3, the optimal inoculation amount for fermentation of all three kinds of cod offal was 4%.

2. Influence of feed liquid ratio on content of polypeptide produced by fermenting cod leftovers

Taking 20g of pasteurized fish skin, respectively setting the material-liquid ratio to be 1:1, 1:3, 1:5, 1:7 and 1:9, wherein the inoculum size is 6%, the temperature is 30 ℃, the rotating speed is 180r/min, carrying out shake culture, fermenting for 24h, centrifuging at 4000r/min after the fermentation is finished, and measuring the peptide content in the supernatant. The above experimental procedures were repeated for fish and fishbone, respectively.

As shown in FIG. 4, the optimal fermentation liquor ratio of all three kinds of cod leftovers is 1: 1.

3. Influence of fermentation time on content of polypeptide produced by fermenting cod leftovers

Taking 20g of pasteurized fish skin, wherein the material-liquid ratio is 1:5, the inoculum size is 6%, the temperature is 30 ℃, the shaking table culture is carried out at the rotating speed of 180r/min, the fermentation is carried out for 0-72h, samples are taken every 12h, the centrifugation is carried out at 4000r/min, and the peptide content in the supernatant is determined. The above experimental procedures were repeated for fish and fishbone, respectively.

As a result, as shown in FIG. 5, the optimum fermentation time for the skin of cod fish was 24 hours, and the optimum fermentation time for both the bones and the flesh of fish was 36 hours.

4. Orthogonal optimization of inoculum size, feed-liquid ratio and fermentation time

According to the result of the single-factor experiment, L9 (3) is adopted³) And (3) performing orthogonal test, namely performing orthogonal optimization on the fermentation process by taking 3 factors of inoculation amount, feed-liquid ratio and fermentation time as influencing factors and taking peptide content as an index, and determining the optimal condition of Bacillus belief MRS for fermenting the cod leftovers.

As can be seen from tables 2, 4 and 5, respectively, the best protocol for obtaining cod skin polypeptide according to the range analysis is A₁B₁C₂Namely, the inoculation amount of the Bacillus belgii MRS is 3 percent, and the material-liquid ratio is 3:1, fermenting for 24 hours according to the optimal combination A₁B₁C₂A verification test is carried out, and the amount of the polypeptide is 13.42 +/-0.46 g/L.

The optimal scheme of the cod fish meat polypeptide is A₁B₁C₁Namely, the inoculation amount of the Bacillus belgii MRS is 3 percent, and the material-liquid ratio is 3:1, fermenting for 24 hours according to the optimal combination A₁B₁C₁A verification test is carried out, and the measured polypeptide amount is 8.61 +/-0.32 g/L.

The optimal scheme of the cod bone polypeptide is A₃B₁C₂Namely, the inoculation amount of the Bacillus belgii MRS is 3 percent, and the material-liquid ratio is 3:1, fermenting for 36h according to the optimal combination A₃B₁C₂A verification test is carried out, and the amount of the polypeptide is measured to be 6.33 +/-0.26 g/L.

TABLE 1 Fish skin orthogonal test factors and horizon table

TABLE 2 Fish skin orthogonal test analysis table

TABLE 3 orthogonal test factors for fish meat (fishbone) and horizon table

Table 4 fish meat orthogonal test analysis table

TABLE 5 fishbone orthogonal test analysis table

Second, optimization of the process for producing polypeptide by enzymolysis of cod leftovers by alkaline protease

1. Influence of alkaline protease dosage on polypeptide content in enzymolysis product

Taking fermentation products of fish skin, fish meat and fish bone under the optimal fermentation condition, respectively adding protease of 0, 200U/g, 400U/g and 600U/g, adjusting pH to 10, temperature to 55 ℃, rotating speed to 180r/min, and performing enzymolysis for 5h, and then centrifuging at 4000r/min to determine the polypeptide content. The method for measuring the content of the polypeptide is the same as that in example 3.

The results are shown in FIG. 6, for cod skin fermentation according to optimal combination A₁B₁C₂After the fermentation is finished, the addition amount of the alkaline protease is set as a single factor, when the addition amount of the alkaline protease is 200U/g, the highest polypeptide content is measured to be 14.14g/L, and the content is significantly different from other conditions with different enzyme addition amounts (P)<0.05), therefore, the optimum is to select the alkaline protease adding amount of 200U/g; according to the optimal combination A in the fish fermentation process₁B₁C₁After the fermentation is finished, the addition amount of the alkaline protease is set as a single factor, when the addition amount of the alkaline protease is 400U/g, the highest polypeptide content is measured to be 19.62g/L, and the content is significantly different from other conditions with different enzyme addition amounts (P)<0.05), therefore, the optimum is to select the alkaline protease adding amount of 400U/g; according to the optimal combination A in the fermentation process of the fishbone₃B₁C₂After the fermentation is finished, the addition amount of the alkaline protease is set as a single factor, when the addition amount of the alkaline protease is 0, the highest polypeptide content is measured to be 7.93g/L, 400U/g is measured to be 7.35g/L, and no significant difference exists between the two (P)>0.05), the amount of the alkaline protease to be added is preferably 0.

2. Influence of enzymolysis time on polypeptide content in enzymolysis product

Taking fermentation products of fish skin, fish meat and fish bones under the optimal fermentation condition, adding 400U/g protease, carrying out shake culture at 55 ℃ and 180r/min of rotation speed at pH 10 for 0-7h, taking samples every two hours from the third hour, and centrifuging at 4000r/min to determine the polypeptide content.

The results are shown in FIG. 7, for cod skin fermentation according to optimal combination A₁B₁C₂After the fermentation is finished, the fermentation liquid is fermented,setting enzymolysis time as single factor, when the enzymolysis time is 3h, the measured polypeptide content is the highest, 14.09g/L, and has significant difference (P) with other conditions of different time<0.05), so the enzymolysis time of 3 hours is selected as the best; according to the optimal combination A in the fish fermentation process₁B₁C₁After the fermentation is finished, setting the enzymolysis time as a single factor, and when the enzymolysis time is 5 hours, measuring the highest polypeptide content which is 16.95g/L, wherein the polypeptide content is obviously different from other polypeptide content under different time conditions (P)<0.05), so the enzymolysis time of 5 hours is selected as the best; according to the optimal combination A in the fermentation process of the fishbone₃B₁C₂After the fermentation is finished, setting the enzymolysis time as a single factor, adding 400U/g of alkaline protease, and when the enzymolysis time is 3 hours, measuring the highest polypeptide content to be 7.79g/L, and then 0 hour to be 7.57 g/L. But there was no significant difference (P) between the two>0.05), so the enzymolysis time of 0h is selected as the best.

3. Determination of polypeptide content and antioxidant activity thereof under optimal fermentation and enzymolysis conditions

Centrifuging fermentation liquor enzymolysis products of fish skin, fish meat and fish bone to remove thallus and impurities, measuring polypeptide content of supernatant, concentrating part of supernatant, freeze-drying to obtain crude polypeptide powder, respectively configuring the crude polypeptide powder into concentrations of 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL and 50mg/mL, and measuring antioxidant activity.

As can be seen from FIG. 8, the IC of the skin, flesh and bone of fish₅₀Values of 17.1, 19.5, 13.8, respectively; and selecting the fishbone polypeptide with the best clearance for subsequent experiments.

Third, the influence of the codfish leftover polypeptide on the dough property

1. Influence of the addition amount of the fishbone polypeptide powder on the dough property

Taking the prepared cod fish bone polypeptide powder, setting the adding amounts of the cod fish bone polypeptide powder to be 0, 1%, 3% and 5% respectively, mixing the cod fish bone polypeptide powder with flour to obtain mixed powder, adding 50% by volume of water, kneading the powder for 15-20min at the temperature of 25-30 ℃ by using a noodle maker, covering the kneaded dough with a preservative film, and standing the dough for 30min at the environmental temperature of 20-30 ℃.

(1) Effect on dough gelatinization Properties

The gelatinization properties of flour not only affect the appearance of the flour product, but also its texture and mouthfeel.

3.0g of the mixed powder was accurately weighed, added to an aluminum box containing 21.43mL of distilled water, sufficiently mixed and stirred with a rotary paddle, and then placed on a Rapid Viscoanalyzer (RVA) for measurement. The test is initially stirred at 960r/min for 10s until a uniform suspension of the sample is formed, and then the rotation speed of 160r/min is maintained until the test is finished. Keeping the initial temperature in RVA at 50 ℃ for 1min, then heating to 95 ℃ at 12 ℃/min for 2.5min, finally cooling to 50 ℃ at 12 ℃/min for 2min, keeping the whole process for 13min, and recording the peak viscosity, the valley viscosity, the attenuation value, the final viscosity, the retrogradation value, the peak time and the gelatinization temperature of the mixed powder in the gelatinization process.

The effect of different amounts of cod bone polypeptide powder added on the dough gelatinizing properties is shown in fig. 9 and table 8. As can be seen from fig. 9, when 1%, 3%, and 5% of the cod fish bone polypeptide powder was added in comparison with wheat flour, the gelatinization viscosity of the mixed powder decreased with the increase in the amount of the added cod fish bone polypeptide powder.

The specific gelatinization characteristic indexes are shown in Table 8, the attenuation value represents the shearing resistance of the starch paste at high temperature and can reflect the thermal stability of starch gelatinization, and the smaller the attenuation value is, the better the thermal stability of the gelatinization is. As is clear from Table 8, the attenuation value tended to decrease with increasing addition amount, and when the addition amount was 3%, it was significantly different from wheat flour (p < 0.05). Retrogradation value is related to starch rearrangement and product retrogradation, with higher values being more susceptible to retrogradation. As is clear from table 8, the retrogradation value significantly decreased (P <0.05) with increasing addition amount based on wheat flour, and a significant difference (P <0.05) was observed from wheat flour when the addition amount was 3%. Therefore, the amount of 3% is preferably selected, and the thermal stability of the mixed powder is improved and the deterioration can be suppressed to some extent.

TABLE 8 influence of different addition amounts of fishbone polypeptide powder on dough gelatinization properties

(2) Influence on dough rheological Properties

2g of dough was cut with scissors and measured with a rheometer. The diameter of the test probe is 25mm, the distance between the two flat plates is 2mm, and the edge of the clamp is coated with silicone oil to prevent moisture from volatilizing. And (3) uniformly balancing the sample at 25 ℃ for 3min after the probe is pressed down, and recovering the deformation of the sample caused by the extrusion of the probe. The frequency is determined to be 10.0rad/s by dynamic stress scanning, and the stress is 0.01-10 percent. The elastic modulus (storagemodulius, G') and the viscous modulus (losssmodulus, G ") of the samples were measured as a function of the stress.

And (3) frequency scanning test: setting the strain amplitude to be 0.05%, the temperature to be 25 ℃ and the frequency to be 0.1-20Hz, and testing to obtain the change curve of G 'and G' of the sample along with the frequency.

The results are shown in fig. 10, where the dynamic visco-elastic behavior of the different reconstituted flour dough systems, G 'and G "increased with increasing frequency (Hz) with a tendency of first-speed to last-slow, and G' of the dough was greater than G", indicating that each dough was more elastic than viscous and semi-solid, biasingly elastic in nature. The elastic modulus G 'and the viscous modulus G' both decrease with increasing addition of the polypeptide powder. Compared with the control group (wheat dough), the dough added with the polypeptide powder has lower elastic modulus (G ') and viscous modulus (G') than the control group, which shows that the addition of the cod bone peptide powder can destroy the gel network structure in the dough and has influence on the viscosity and the fluidity of the dough.

Temperature scanning test: the sample was heated from 25 ℃ to 95 ℃ at a rate of 5 ℃/min, frequency lHz, strain amplitude 0.05%, and the curve of the relevant modulus of the sample as a function of temperature was recorded.

As can be seen from FIG. 11, the viscoelasticity of the mixed flour was less than that of the control wheat dough during heating, and G 'showed an upward trend as the amount of the cod bone peptide powder added was increased, and G' of each dough were slightly decreased at 25 to 65 ℃, indicating that the dough system was stable at this stage. When the temperature is higher than 65 ℃, the temperature of each dough G 'and G' rapidly rises to about 85 ℃ to reach the maximum viscoelasticity value, the peak viscoelasticity of the mixed flour dough is smaller than that of the control group, and the peak viscoelasticity of the mixed flour dough is gradually increased along with the increase of the addition amount. As the temperature is gradually increased, the starch granules break, resulting in a sharp drop in the G 'and G' of the dough.

In conclusion, the dough with the addition amount of 3 percent of the fishbone polypeptide powder is pressed into noodles, and the quality of the noodles is the best.

Influence of cod leftover polypeptide on noodle quality

The cod leftover polypeptide powder is made into dough by adding 3 percent of the polypeptide powder, and then the dough is pressed into noodles, and the water absorption rate and the breaking rate of the noodles, the texture characteristics of the noodles and the tensile characteristics of the noodles are tested.

1. Noodle water absorption rate and noodle breakage rate

Water absorption: accurately weighing 20g of noodles, putting the noodles into 500mL of distilled water, boiling for 3-5min, taking out, flushing with 50mL of distilled water, collecting water for boiling the noodles and flushing, draining for 5min at room temperature, accurately weighing, and calculating according to the formula:

noodle water absorption (%) (W)₂-W₁)/W₁×100％；

Wherein, W₁: weight of noodle before cooking, g; w₂: weight of cooked back noodles, g.

The broken strip rate: taking 20 noodles, putting into 500mL of boiling water, boiling for 3-5min, and calculating the noodle breaking rate.

The water absorption rate is an important factor for evaluating the cooking characteristics of the noodles, reflects the content of the gluten protein, and indicates that the higher the value is, the tougher the noodles are. The influence of the addition amount of 3% of the polypeptide powder on the water absorption of the noodles is shown in table 9, and the water absorption of the noodles to which the polypeptide powder was added was decreased as compared with the control group, indicating that the noodles were more chewy and the quality of the noodles was improved to some extent. The noodle containing 3% of the polypeptide powder had an increased noodle breakage rate compared to the control group, indicating that the noodle had decreased viscoelasticity and strength.

TABLE 93% influence of the amount of polypeptide powder added on Water absorption and strip breakage of noodle

2. Texture characteristics of noodles

Weighing 20g of noodles, putting into 500mL of boiling water, boiling for 3-5min, taking out, draining for 5min at room temperature, measuring by using a texture analyzer, measuring each group of samples for 3 times, and taking an average value.

The texture of the noodles is closely related to the shape and taste of the noodles in sensory indexes. The influence of the addition of 3% of the polypeptide on the texture indexes such as hardness, elasticity, chewiness of the noodles is shown in table 10, and the hardness of the noodles to which the polypeptide powder is added is significantly reduced (p <0.05) as compared with the control group, and the stickiness and elasticity of the noodles to which the polypeptide powder is added are significantly increased (p <0.05) as compared with the control group, which indicates that the cohesiveness of the noodles to which the polypeptide powder is added is enhanced, the relative hardness is reduced, the elasticity is enhanced, the noodles are glossy, smooth and chewy, and the flavor and nutrition of the noodles are increased.

Influence of the amount of polypeptide added in Table 103% on texture characteristics of noodles

(3) Noodle stretch characteristics

Weighing 20g of noodle, boiling in boiling water for 3-5min, taking out, rinsing with clear water for 30s, performing tensile test with intelligent electronic tension meter, measuring elongation, breaking force and tensile strength of noodle, measuring each group of samples for 3 times, and taking average value.

The tensile properties of the noodles were tested to simulate the human chewing process. The influence of the addition amount of the polypeptide of 3% on the texture characteristics of the noodles is shown in table 11, and the higher the breaking force, the higher the toughness of the noodles. The addition of the fishbone polypeptide powder has no significant difference on the breaking force (p > 0.05). The elongation and the stretch-breaking distance of the noodles to which the polypeptide powder was added were significantly increased (p <0.05) compared to the control group. Therefore, the addition of the fishbone polypeptide powder improves the toughness of the noodles, makes the noodles more chewy and improves the quality of the noodles.

Effect of the amount of polypeptide added in Table 113% on tensile Properties of noodles

Example 4: safety of cod leftover peptide powder

The method aims to judge the safety of the fish bone polypeptide powder for human administration and provide a basis for toxicological safety for later edible safety. Acute toxicity test, Ames test and oral administration mode in national food safety standard are adopted for 90d feeding test. Fishbone peptide powder mice orally received a Maximum Tolerated Dose (MTD) >1.00g/kg · BW; the Ames test result is negative.

After the mice are fed for 90 days, the food intake and the food utilization rate of the high-dose group of the tested sample are different from those of the normal control group, and other indexes have no significant difference; the indexes of the SD mice in the low and medium dose groups, such as body weight, food intake, food utilization rate, hematology, blood biochemistry, organ weight, organ body ratio and the like, have no significant difference compared with the normal control group. No biologically significant changes were observed in the coefficients of the major organs. The existing test results prove that the fishbone peptide powder is safe and reliable to use by human body in the range of less than 1.0g/kg (BW).

1. Acute toxicity test

According to the normal eating amount of human, assuming that the noodles are eaten three times a day, 300g (dry weight of noodles) of each time is the maximum eating amount, and according to the optimal addition amount of 3% of the fishbone polypeptide powder, designing a mouse acute experiment, wherein mice are divided into four groups, namely a blank group, a low-dose group, a medium-dose group and a high-dose group, and each group comprises 12 mice. The blank mice are perfused with 0.2mL of physiological saline every time, the low dose mice are perfused with 0.005g (polypeptide powder)/10 g (BW), the medium dose mice are perfused with 0.010g (polypeptide powder)/10 g (BW), the high dose mice are perfused with 0.025g (polypeptide powder)/10 g (BW), and the perfusing amount is 0.2 mL. Gavage for 14d, during which the growth of the mice is observed; after 14 days, the mice were sacrificed by cervical dislocation.

During the feeding period, the diet and activity of the test mice are normal, one mouse in the high-dose group dies, and other groups do not have any severe symptoms and death phenomenon. 12h before sacrifice, water is not cut off when food is cut off, organs such as liver, kidney, spleen, pancreas and the like are dissected and taken, and compared with blank organs, the organs are observed by naked eyes to be abnormal, which shows that the maximum oral tolerance dose of the fish bone peptide powder mice is more than 1.0g/kg & BW.

2. Ames test

The plate selection and incorporation method uses qualified Salmonella typhimurium mutant strains TA97, TA98, TA100 and TA102, and 5 dose groups of 8, 40, 200, 1000 and 5000 mug/dish, an untreated control group, a solvent control group and a positive control group are cultured for 48h at 37 ℃.

Under the condition of adding S9 and not adding S9, the detection is repeated twice under the same experimental conditions, the number of the retrovariant colonies of each dose group does not exceed 2 times of that of an untreated control group, and the number of the retrovariant colonies of a positive control group exceeds 2 times of that of the untreated control group, so that the fishbone peptide powder has no mutagenic effect on the salmonella typhimurium and has a negative result.

3. 90d feeding test

Animals were fed in a single cage, were fed freely and were fed water, and the activity and growth of the experimental animals were observed daily. Respectively feeding the feed containing 3 percent, 5.0 percent, 10.0 percent and the feed without the polypeptide powder for 90 days continuously. The weight and the feed amount were weighed once a week, and the food intake and food remaining were recorded. Calculate weekly and total weight gain, feed intake and food utilization. And (4) blood sampling is carried out in the middle stage of the experiment and after the experiment is finished to measure hemoglobin, erythrocyte count and leucocyte count and classification. After the experiment is finished, mice of each dose group are anesthetized, eyeballs are picked out, blood is taken, and serum is separated to determine serum biochemical indexes. The animals were dissected and the visceral changes were visually observed.

During the experiment, each group of animals has flexible action, quick response, clean and tidy quilt, no secretion in eyes, noses and mouths, normal eating and drinking, good growth and development, no abnormal behavior and toxic symptoms and no death. Actual food intake per week and average body weight per week were calculated by 13 weeks of feeding. Through analysis, mice in each dose group have no significant difference in initial weight, weight increment and comparison with a control group (p > 0.05). The difference among the groups has no dose response relationship and has no statistical significance, which indicates that the cod polypeptide powder has no obvious influence on the growth and development of mice. Compared with a control group, the results of measuring various conventional hematology indexes (hemoglobin, erythrocyte count, leucocyte count and classification) of the blood of each dose group by tail blood collection at the middle stage of the experiment and before the end of the experiment of each dose group have no significant difference (p is more than 0.05). The result values of serum total cholesterol, creatinine, triglyceride, blood sugar, aspartate aminotransferase, alanine aminotransferase, urea nitrogen, total protein, albumin and alkaline phosphatase in each dose group of mice were not significantly different from those in the control group (p > 0.05). The weight of the liver, the kidney, the spleen and the testis, the liver body ratio, the kidney body ratio, the spleen body ratio and the testis body ratio of the mice in each dose group have no statistical significance compared with the solvent control group (p is more than 0.05). The animals of each dose group are dissected and generally observed, and organs in the thoracoabdominal cavity such as the liver, the kidney, the spleen, the lung, the stomach and the intestine of the male and female mice of each experimental dose group are normal in appearance color and organ size compared with the normal control group, and no pathological change with practical significance is found.

In conclusion, the fish bone polypeptide powder is verified to completely meet the safety of human body administration and is non-toxic and harmless.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims

1. Nutritional noodles added with fish polypeptide powder are characterized by comprising fish polypeptide powder and flour.

2. The nutritional noodles according to claim 1, wherein the fish polypeptide powder is obtained by fermenting fish leftovers with Bacillus belgii and then subjecting the fish leftovers to enzymatic hydrolysis with alkaline protease.

3. Nutritional noodles according to claim 2, wherein Bacillus belgii is Bacillus belgii MRS with the accession number CGMCC No. 18065.

4. The method for preparing nutritious noodles according to claim 1, characterized in that the preparation method comprises the steps of:

(1) after activation of the Bacillus beleisi, performing shake culture in an MRS liquid culture medium at the temperature of 28-30 ℃ and at the speed of 150-180rpm/min for 24-48 h to prepare a Bacillus beleisi seed liquid;

(2) crushing the fish leftovers by using a crusher, adding distilled water for sterilization, cooling to room temperature to obtain fish leftover slurry, adding the Bacillus beiLeisi seed liquid, and fermenting at the temperature of 28-30 ℃ and 180rpm/min for 24-36h at 150-30 ℃ to obtain a fish fermentation product;

(3) performing enzymolysis on the fish fermentation product for 3-5h at the pH of 10, 55-58 ℃ and 180rpm/min by using alkaline protease, centrifuging the fish enzymolysis liquid, taking supernatant, concentrating, and freeze-drying to obtain fish polypeptide powder;

(4) mixing the fish polypeptide powder with flour and water, kneading with noodle machine for 15-20min, standing for 20-30min while maintaining humidity, pressing into noodles with noodle press, dewatering, and drying to obtain nutritional noodles.

5. The method according to claim 4, wherein the concentration of the Bacillus belgii seed solution in the step (1) is 6 to 8X 10⁷cfu/mL。

6. The preparation method according to claim 4, wherein the feed-liquid ratio of the fish leftovers crushed in the step (2) to the distilled water is 1:1-3: 1.

7. The method according to claim 4, wherein the Bacillus belgii seed solution is added in an amount of 3-4% by mass based on the fish offal slurry in the step (2).

8. The method according to claim 4, wherein the amount of alkaline protease added in step (3) is 200-400U/g.

9. The preparation method according to claim 4, wherein the adding amount of the fish polypeptide powder in the step (4) is 3-5% of the mass of the flour, and the adding amount of the water is 40-50% of the mass of the flour.

10. Nutritional noodles according to claim 2 or a preparation method according to claim 4, wherein the fish offal comprises at least one of fish skin, fish meat, fish bone.