Disclosure of Invention
In view of the above, the present invention aims to provide a hydrolysate prepared from carp scales and having anti-freezing activity, an anti-freezing agent, and applications thereof, wherein the hydrolysate having anti-freezing activity has high thermal hysteresis activity.
The invention provides a hydrolysate with anti-freezing activity prepared from carp scales, which is prepared by the following steps:
1) removing foreign proteins and pigments from the carp scales, and removing calcium components to obtain pretreated carp scales;
2) carrying out enzymolysis on the pretreated carp scales under the action of alkaline protease, and inactivating enzyme to obtain an enzymolysis liquid;
the temperature of enzymolysis is 50-60 ℃, the pH value of an enzymolysis system is 7-9, and the enzymolysis time is 4-6 h.
Preferably, the enzyme activity of the alkaline protease in the step 2) is more than or equal to 200000U/g.
Preferably, the adding mass of the alkaline protease is 0.75-0.85% of the total mass of the carp scales.
Preferably, the method for removing the foreign proteins and pigments from the carp scales in the step 1) comprises the steps of treating the carp scales with a strong alkaline solution and washing the carp scales with water to be neutral.
Preferably, the mass volume ratio of the carp scales to the strong alkali solution is 1g: (10-15) mL.
Preferably, the molar concentration of the strong alkali solution is 0.08-0.12 mol/L.
Preferably, the temperature for treating the carp scales by using a strong alkaline solution is 20-25 ℃;
the time for treating the carp scales by using the strong alkali solution is 20-24 hours;
preferably, the carp scales are treated by using a strong alkali solution along with oscillation; the rotating speed of the oscillation is 200-4000 rpm.
Preferably, the method for decalcifying the carp scales from which the foreign proteins and pigments are removed in the step 1) comprises treating the carp scales with an inorganic acid aqueous solution, and washing the carp scales with water to be neutral.
The invention provides an antifreeze agent derived from carp scales, which comprises a hydrolysate with antifreeze activity.
The invention provides the application of the hydrolysate with anti-freezing activity or the anti-freezing agent in food processing or medicine production.
The invention provides a hydrolysate prepared from carp scales and having anti-freezing activity, which is prepared by taking the carp scales as raw materials, firstly, foreign proteins and pigments of the fish scales are removed, the interference of the foreign proteins in the enzymolysis process is reduced, and the content of anti-freezing peptides in the hydrolysate is increased; then alkaline protease is used for enzymolysis, and the anti-freezing activity of collagen in the fish scales can be retained to the maximum extent. The hydrolysate with anti-freezing activity provided by the invention has the thermal hysteresis activity of more than 0.8, and has stronger protective effect on protein denaturation in the freezing process.
Meanwhile, the hydrolysate with the anti-freezing activity provided by the invention takes the carp scales as raw materials, can solve the problem of utilization of waste carp scales in processing the carp, improves the added value of the carp leftovers, changes waste into valuable, and improves the comprehensive utilization rate of the carp scales. The hydrolysate with the anti-freezing activity provided by the invention has the advantages of simple preparation process, mild conditions, low production cost and high equipment utilization rate, and is very suitable for industrial mass production.
The invention further defines a pretreatment process for removing foreign proteins and pigments and decalcifying. Before enzymolysis of the fish scales, strong alkali is used for removing foreign protein and pigment of the fish scales, interference of the foreign protein in the enzymolysis process is reduced, and the content of antifreeze peptide in hydrolysate is increased. Meanwhile, the hydroxyapatite layer on the surface of the fish scales and the hydroxyapatite in the fish scales are removed by using inorganic acid before enzymolysis, so that the contact area between enzyme and collagen is increased in the enzymolysis process, the hydrolysis efficiency is improved, the hydrolysis time is shortened, and the content of salt in hydrolysate is lower due to the removal of the hydroxyapatite.
Detailed Description
The invention provides a hydrolysate with anti-freezing activity prepared from carp scales, which is prepared by the following steps:
1) removing foreign proteins and pigments from the carp scales, and removing calcium components to obtain pretreated carp scales;
2) carrying out enzymolysis on the pretreated carp scales under the action of alkaline protease, and inactivating enzyme to obtain an enzymolysis liquid;
the temperature of enzymolysis is 50-60 ℃, the pH value of an enzymolysis system is 7-9, and the enzymolysis time is 4-6 h.
The invention removes foreign protein and pigment from the carp scale, removes calcium component, and obtains the pretreated carp scale.
In the present invention, the method for removing foreign proteins and pigments from the carp scales preferably comprises treating the carp scales with a strong alkaline solution and washing the carp scales with water to be neutral. The mass volume ratio of the carp scales to the strong alkali solution is preferably 1g: (10-15) mL, more preferably 1g: (11-14) mL, more preferably 1g: (12-13) mL. The molar concentration of the strong alkali solution is preferably 0.08-0.12 mol/L, and more preferably 0.1 mol/L. The temperature for treating the carp scales by using the strong alkali solution is preferably 20-25 ℃, more preferably 21-24 ℃, and more preferably 22-23 ℃. The time for treating the carp scales by using the strong alkali solution is preferably 20-24 hours, more preferably 21-23 hours, and most preferably 22 hours. Preferably, the carp scales are treated by using a strong alkali solution along with oscillation; the rotation speed of the oscillation is preferably 200 to 4000rpm, more preferably 400 to 3000rpm, further preferably 500 to 1000rpm, and most preferably 800 rpm. The strong alkaline solution preferably comprises sodium hydroxide and potassium hydroxide.
In the present invention, the method for decalcifying carp scales from which foreign proteins and pigments are removed preferably comprises treating carp scales with an aqueous solution of an inorganic acid, and washing the carp scales with water to neutrality. The mass volume ratio of the carp scales to the inorganic acid aqueous solution is preferably 1g (12-17) mL, more preferably 1g:15 mL. The concentration of the inorganic acid aqueous solution is preferably 0.1 to 1mol/L, and more preferably 0.5 mol/L. The method for treating the carp scales by using the inorganic acid aqueous solution is the same as the method for treating the carp scales by using strong alkali, and details are not repeated herein.
After the pretreated carp scales are obtained, the pretreated carp scales are preferably dried and crushed by the method. The method of drying and pulverizing is not particularly limited in the present invention, and a drying and pulverizing scheme well known in the art may be employed.
After the pretreated carp scales are obtained, the pretreated carp scales are subjected to enzymolysis under the action of alkaline protease, and enzyme deactivation is carried out to obtain an enzymolysis solution containing a hydrolysate with anti-freezing activity.
In the invention, the enzyme activity of the alkaline protease is more than or equal to 200000U/g. The adding mass of the alkaline protease is preferably 0.75-0.85% of the total mass of the carp scales, and more preferably 0.8%. The temperature of enzymolysis is preferably 52-58 ℃, and more preferably 55 ℃. The pH value of the enzymolysis system is preferably 8. The enzymolysis time is preferably 4.5-5.5 h, and more preferably 5 h.
The method for inactivating the enzyme is not particularly limited in the present invention, and an enzyme inactivation scheme well known in the art, for example, heating with boiling water, may be used.
After obtaining an enzymatic hydrolysate containing a hydrolysate having anti-freeze activity, it is preferably centrifuged and dried. And centrifuging, collecting supernatant, and freeze-drying the supernatant to obtain the fish scale hydrolysate. The rotating speed of the centrifugation is preferably 7000-9000 rpm, more preferably 8000rpm, and the time of the centrifugation is preferably 12-17 min, more preferably 15 min. The centrifugation temperature is preferably 2-6 ℃, and more preferably 4 ℃.
In the invention, the antifreeze activity of the prepared fish scale hydrolysate is also detected. The assay includes thermal hysteresis activity assay and protection against protein freeze denaturation during freeze-thaw cycles. The thermal hysteresis activity is preferably detected by differential scanning calorimetry. Protection against freeze denaturation of proteins during the freeze-thaw cycle was verified by measuring the residual activity of catalase after the freeze-thaw cycle. Experimental results show that the hysteresis activity of the hydrolysate with the anti-freezing activity is more than 0.8, the hydrolysate has a strong protection effect on protein denaturation in a freezing process, and the residual activity of catalase is more than 80%.
Based on the fact that the hydrolysate has strong anti-freezing activity, the invention provides an anti-freezing agent derived from carp scales, which comprises the hydrolysate with anti-freezing activity. The invention has no special limitation on the dosage form of the antifreeze, and the antifreeze which is well known in the field can be adopted. The proportion of the hydrolysate having anti-freeze activity is not particularly limited in the present invention, and it is sufficient to refer to the content of active ingredients in the anti-freeze agent well known in the art.
The invention provides the application of the hydrolysate with anti-freezing activity or the anti-freezing agent in food processing or medicine production.
The hydrolysate having anti-freeze activity prepared from carp scales, the anti-freeze agent and the application thereof provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention
Example 1
A method for preparing hydrolysate with anti-freeze activity from carp scale
(1) Removing foreign proteins and pigments of fish scales: adding 0.1mol/L sodium hydroxide solution into 0.5kg of carp scales according to the material-liquid ratio of 1g:10mL, stirring at 25 ℃ for 24h at the stirring speed of 200rmp, then washing with running water for 2h, and detecting the pH of a washing liquid to be neutral;
(2) fish scale decalcification: adding 0.5mol/L hydrochloric acid aqueous solution into the fish scales with foreign proteins and pigments removed according to the feed-liquid ratio of 1g:10mL, stirring at 25 ℃ for 24h at the stirring speed of 200rmp, then washing with running water for 2h, and detecting the pH of a washing solution to be neutral;
(3) drying and crushing: drying the decalcified fish scales at 35 ℃, and crushing to obtain fish scale powder;
(4) enzymolysis of fish scales: adding pure water into the fish scale powder according to the feed-liquid ratio of 1g to 20mL, adding alkaline protease with the enzyme activity of 200000U/g, wherein the addition amount of the alkaline protease is 0.75 percent of the mass of the fish scale powder, hydrolyzing for 4h at the temperature of 50 ℃, and maintaining the pH value of an enzymolysis system to be 8 by using 1mol/L sodium hydroxide solution.
(5) Heating to inactivate enzyme, namely heating the enzyme system subjected to the enzyme reaction for 15min by adopting a boiling water bath to inactivate the enzyme, and then cooling the enzyme system to room temperature by using running water.
(6) Centrifugal drying: centrifuging the enzyme system at 4 ℃ for 15min, collecting supernatant, wherein the centrifugation speed is 8000rmp, and freeze-drying the supernatant to obtain the fish scale hydrolysate.
And weighing the mass of the scale hydrolysate, and calculating the yield of the scale hydrolysate according to the formula (1).
Fish scale hydrolysate yield (%) ═ mass of fish scale hydrolysate/mass of carp scale × 100% formula (1)
And measuring the thermal hysteresis activity of the fish scale hydrolysate by adopting differential scanning calorimetry. The thermal hysteresis activity detection method comprises the following specific steps: THA assay reference Cao et al[1]The method of (3) is slightly modified. And dissolving the hydrolysate after freeze-drying in deionized water to prepare a solution with the concentration of 20 mg/mL. A5 uL portion of the solution was sealed in a crucible, and the sealed crucible was placed in a DSC apparatus to measure the THA of the hydrolysate. Setting the initial temperature to 20 ℃, cooling to-20 ℃ at the speed of 5 ℃/min, and keeping for 2 min; then the temperature is raised to 20 ℃ at the same speed, and the temperature is kept for 2min, so that the melting curve of the hydrolysate is obtained. Then cooling to-20 deg.C again at the same rate, maintaining for 2min, and heating at 1 deg.C/min until the solution is partially molten, and the temperature at this moment is called retention temperature (T)h) The temperature was maintained for 2min and finally the temperature was raised to 20 ℃ at a rate of 1 ℃/min. Continuously repeating the above processes, selecting different Th until THA obtains the maximum value, and calculating THA according to formula (2):
THA=Th-T0 (2)
wherein THA is thermal hysteresis activity, DEG C; t ishRefers to the retention temperature, deg.C; t0 refers to the onset crystallization temperature, deg.C.
The protection effect on catalase in the freeze-thaw cycle is adopted to reflect the inhibition effect of the fish scale hydrolysate on protein denaturation at low temperature. Protection effect on catalase in freeze-thaw cycle with reference to Lixiankun[1]The method of (3) is slightly modified. Dissolving the lyophilized hydrolysate in deionized water to prepare a hydrolysate solution with the concentration of 2mg/mL, and mixing the hydrolysate solution with a catalase solution diluted by 500 times in an equal volume to obtain a catalase solution with the hydrolysate concentration of 1mg/mL and diluted by 1000 times, wherein the blank group is the catalase solution without the hydrolysate and diluted by 1000 times. Then the two enzyme solutions are put into a temperature of minus 20 ℃ for freezing for 24h at minus 20 ℃ and 12h at 4 ℃, the freeze-thaw cycle is repeated for 4 times, and the initial activity of the hydrogen peroxide enzyme and the activity of the hydrogen peroxide enzyme after 4 freeze-thaw cycles are measured. The determination method comprises the following steps: 200uL of sample was added to 1500. mu.L of 0.1M PBS buffer pH 7.0 and 300. mu.L of 0.1M hydrogen peroxide solution at A240Measuring absorbance, and observing the absorbance within 4minRecording the absorbance once every 1min according to the change of the luminosity, and calculating the activity of the peroxidase according to a formula (3) and a formula (4):
ΔA240=A0-A1 (3)
in the formula, A0Refers to the absorbance of the control tube to which the inactivated enzyme was added; a. the1Absorbance of sample tube.
In the formula, VtRefers to the total volume of the sample solution, mL; vsRefers to the volume of sample solution for determination, mL; t refers to the time from the addition of hydrogen peroxide to the last recording, min; w refers to the mass of enzyme in the sample solution, g. See prior art ([1 ]].Cao H,Zhao Y,Zhu Y B,et al.Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin[J].Food Chemistry,2016,194(MAR.1):1245-1253.[2]Preparation of antifreeze polypeptide from pigskin gelatin and cryo-protection study [ D]University of Fuzhou, 2013:31-39.
The results show that the calculated fish scale hydrolysate yield is 68%, the thermal hysteresis activity is 0.8 ℃, the catalase residual activity after adding the hydrolysate is 80%, and the catalase residual activity of the blank group without adding the hydrolysate is 35%.
Example 2
A method for preparing hydrolysate with anti-freeze activity from carp scale
(1) Removing impure proteins and pigments of fish scales, namely adding 0.1mol/L sodium hydroxide solution into 0.5kg of carp scales according to the material-liquid ratio of 1g to 15mL, stirring at 23 ℃ for 22h at the stirring speed of 500rmp, then washing with running water for 2h, and detecting the pH value of a washing liquid to be neutral;
(2) fish scale decalcification: adding 0.5mol/L hydrochloric acid aqueous solution into the fish scales with foreign proteins and pigments removed according to the feed-liquid ratio of 1g:15mL, stirring at the temperature of 23 ℃ for 22 hours at the stirring speed of 500rmp, then washing with running water for 2 hours, and detecting the pH of a washing liquid to be neutral;
(3) drying and crushing: drying the decalcified fish scales at 35 ℃, and crushing to obtain fish scale powder;
(4) enzymolysis of fish scales: adding pure water into the fish scale powder according to the feed-liquid ratio of 1g to 20mL, adding alkaline protease with the enzyme activity of 200000U/g, wherein the addition amount of the alkaline protease is 0.8 percent of the weight of the fish scale powder, hydrolyzing for 5h at the temperature of 58 ℃, and maintaining the pH value of an enzymolysis system to be 8 by using 1mol/L sodium hydroxide solution.
(5) Heating to inactivate enzyme, namely heating the enzyme system subjected to the enzyme reaction for 15min by adopting a boiling water bath to inactivate the enzyme, and then cooling the enzyme system to room temperature by using running water.
(6) Centrifugal drying: centrifuging the enzyme system at 4 ℃ for 15min, collecting supernatant, wherein the centrifugation speed is 8000rmp, and freeze-drying the supernatant to obtain the fish scale hydrolysate.
The yield and the anti-freeze activity of the fish scale hydrolysate were tested according to the method of example 1. The results show that the calculated fish scale hydrolysate yield is 71%, the thermal hysteresis activity is 0.9 ℃, the catalase residual activity after adding the hydrolysate is 83%, and the catalase residual activity of the blank group without adding the hydrolysate is 35%.
Example 3
A method for preparing hydrolysate with anti-freeze activity from carp scale
(1) Removing impure proteins and pigments of fish scales, namely adding 0.1mol/L sodium hydroxide solution into 0.5kg of carp scales according to the material-liquid ratio of 1g to 15mL, stirring at the temperature of 20 ℃ for 21h at the stirring speed of 1000rmp, then washing for 2h with running water, and detecting the pH value of a washing solution to be neutral;
(2) fish scale decalcification: adding 0.5mol/L hydrochloric acid aqueous solution into the fish scales with foreign proteins and pigments removed according to the feed-liquid ratio of 1g:15mL, stirring at the temperature of 20 ℃ for 21h at the stirring speed of 1000rmp, then washing with running water for 2h, and detecting the pH of a washing solution to be neutral;
(3) drying and crushing: drying the decalcified fish scales at 35 ℃, and crushing to obtain fish scale powder;
(4) enzymolysis of fish scales: adding pure water into the fish scale powder according to the feed liquid ratio of 1g to 20mL, adding alkaline protease with the enzyme activity of 200000U/g, wherein the addition amount of the alkaline protease is 0.78% of the weight of the fish scale powder, hydrolyzing for 5h at the temperature of 60 ℃, and maintaining the pH value of an enzymolysis system to be 8 by using 1mol/L sodium hydroxide solution.
(5) Heating to inactivate enzyme, namely heating the enzyme system subjected to the enzyme reaction for 15min by adopting a boiling water bath to inactivate the enzyme, and then cooling the enzyme system to room temperature by using running water.
(6) Centrifugal drying: centrifuging the enzyme system at 4 ℃ for 15min, collecting supernatant, wherein the centrifugation speed is 8000rmp, and freeze-drying the supernatant to obtain the fish scale hydrolysate.
The yield and the anti-freeze activity of the fish scale hydrolysate were tested according to the method of example 1. The calculated fish scale hydrolysate yield is 70%, the thermal hysteresis activity is 0.88 ℃, the catalase residual activity of the added hydrolysate is 82%, and the catalase residual activity of the blank group without the added hydrolysate is 35%.
Comparative example 1
A method for preparing hydrolysate with anti-freeze activity from carp scale
(1) Removing impure proteins and pigments of fish scales, namely adding 0.1mol/L sodium hydroxide solution into 0.5kg of carp scales according to the material-liquid ratio of 1g to 10mL, stirring at 23 ℃ for 22h at the stirring speed of 500rmp, then washing with running water for 2h, and detecting the pH value of a washing liquid to be neutral;
(2) fish scale decalcification: adding 0.5mol/L hydrochloric acid aqueous solution into the fish scales with foreign proteins and pigments removed according to the feed-liquid ratio of 1g:10mL, stirring at 23 ℃ for 22h at the stirring speed of 500rmp, then washing with running water for 2h, and detecting the pH of a washing solution to be neutral;
(3) drying and crushing: drying the decalcified fish scales at 35 ℃, and crushing to obtain fish scale powder;
(4) enzymolysis of fish scales: adding pure water into the fish scale powder according to the feed-liquid ratio of 1g to 20mL, adding flavourzyme with the enzyme activity of 300000U/g, wherein the addition amount of the flavourzyme is 0.75 percent of the weight of the fish scale powder, hydrolyzing for 5 hours at the temperature of 50 ℃, and maintaining the pH value of an enzymolysis system to be 7 by using 1mol/L sodium hydroxide solution.
(5) Heating to inactivate enzyme, namely heating the enzyme system subjected to the enzyme reaction for 15min by adopting a boiling water bath to inactivate the enzyme, and then cooling the enzyme system to room temperature by using running water.
(6) Centrifugal drying: centrifuging the enzyme system at 4 ℃ for 15min, collecting supernatant, wherein the centrifugation speed is 8000rmp, and freeze-drying the supernatant to obtain the fish scale hydrolysate.
The yield and the anti-freeze activity of the fish scale hydrolysate were tested according to the method of example 1. The results show that the calculated fish scale hydrolysate yield is 50%, the thermal hysteresis activity is 0.32 ℃, the catalase residual activity after adding the hydrolysate is 42%, and the catalase residual activity of the blank group without adding the hydrolysate is 35%.
Comparative example 2
A method for preparing hydrolysate with anti-freeze activity from carp scale
(1) Removing impure proteins and pigments of fish scales, namely adding 0.1mol/L sodium hydroxide solution into 0.5kg of carp scales according to the material-liquid ratio of 1g to 10mL, stirring at 23 ℃ for 22h at the stirring speed of 500rmp, then washing with running water for 2h, and detecting the pH value of a washing liquid to be neutral;
(2) fish scale decalcification: adding 0.5mol/L hydrochloric acid aqueous solution into the fish scales with foreign proteins and pigments removed according to the feed-liquid ratio of 1g:10mL, stirring at 23 ℃ for 22h at the stirring speed of 500rmp, then washing with running water for 2h, and detecting the pH of a washing solution to be neutral;
(3) drying and crushing: drying the decalcified fish scales at 35 ℃, and crushing to obtain fish scale powder;
(4) enzymolysis of fish scales: adding pure water into the fish scale powder according to the feed-liquid ratio of 1g to 20mL, adding neutral protease with the enzyme activity of 100000U/g, wherein the addition amount of the neutral protease is 0.75 percent of the weight of the fish scale powder, hydrolyzing for 4h at the temperature of 50 ℃, and maintaining the pH value of an enzymolysis system to be 7 by using 1mol/L sodium hydroxide solution.
(5) Heating to inactivate enzyme, namely heating the enzyme system subjected to the enzyme reaction for 15min by adopting a boiling water bath to inactivate the enzyme, and then cooling the enzyme system to room temperature by using running water.
(6) Centrifugal drying: centrifuging the enzyme system at 4 ℃ for 15min, collecting supernatant, wherein the centrifugation speed is 8000rmp, and freeze-drying the supernatant to obtain the fish scale hydrolysate.
The yield and the anti-freeze activity of the fish scale hydrolysate were tested according to the method of example 1. The results show that the calculated fish scale hydrolysate yield is 65%, the thermal hysteresis activity is 0.7 ℃, the catalase residual activity after adding the hydrolysate is 51%, and the catalase residual activity of the blank group without adding the hydrolysate is 35%.
Comparative example 3
A method for preparing hydrolysate with anti-freeze activity from carp scale
(1) Removing foreign proteins and pigments of fish scales: adding 0.1mol/L sodium hydroxide solution into 0.5kg of carp scales according to the material-liquid ratio of 1g:15mL, stirring at 23 ℃ for 22h at the stirring speed of 500rmp, then washing with running water for 2h, and detecting the pH of a washing solution to be neutral;
(2) fish scale decalcification: adding 0.5mol/L hydrochloric acid aqueous solution into the fish scales with foreign proteins and pigments removed according to the feed-liquid ratio of 1g:15mL, stirring at the temperature of 23 ℃ for 22 hours at the stirring speed of 500rmp, then washing with running water for 2 hours, and detecting the pH of a washing liquid to be neutral;
(3) drying and crushing: drying the decalcified fish scales at 35 ℃, and crushing to obtain fish scale powder;
(4) enzymolysis of fish scales: adding pure water into the fish scale powder according to the feed liquid ratio of 1g to 20mL, adding alkaline protease with the enzyme activity of 200000U/g, wherein the addition amount of the alkaline protease is 0.75 percent of the weight of the fish scale powder, hydrolyzing for 2h at the temperature of 50 ℃, and maintaining the pH value of an enzymolysis system to be 8 by using 1mol/L sodium hydroxide solution.
(5) Heating to inactivate enzyme, namely heating the enzyme system subjected to the enzyme reaction for 15min by adopting a boiling water bath to inactivate the enzyme, and then cooling the enzyme system to room temperature by using running water.
(6) Centrifugal drying: centrifuging the enzyme system at 4 ℃ for 15min, collecting supernatant, wherein the centrifugation speed is 8000rmp, and freeze-drying the supernatant to obtain the fish scale hydrolysate.
The yield and the anti-freeze activity of the fish scale hydrolysate were tested according to the method of example 1. The results showed that the calculated yield of the fish scale hydrolysate was 42%, the thermal hysteresis activity was 0.15 ℃, the residual catalase activity after adding the hydrolysate was 25.0%, and the residual catalase activity of the blank group without adding the hydrolysate was 21%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.