KR20240081828A - Manufacturing method of feed additive obtained from tuna frame meat for improving palatability and antioxidant capacity of pet food and feed additive obtained from tuna frame meat thereof - Google Patents

Abstract

The present invention relates to the production of a feed additive that improves the palatability of pet food and pet food using tuna frame meat and provides antioxidant properties. More specifically, it relates to producing a protein hydrolyzed extract of tuna frame meat, Accordingly, the Maillard reaction is induced through the process of adding and heating lactic acid bacteria fermentation and reducing sugar, ultimately improving the flavor and antioxidant capacity through lactic acid bacteria fermentation and adding reducing sugar, thereby improving the palatability and antioxidant properties of pet food and snacks (pet food). It relates to a method of manufacturing a pet food additive that promotes

Description

Method for manufacturing a lactic acid bacteria-fermented feed additive derived from tuna frame meat to improve the antioxidant capacity of pet food, and a lactic acid bacteria-fermented feed additive derived from tuna frame meat manufactured using the same CAPACITY OF PET FOOD AND FEED ADDITIVE OBTAINED FROM TUNA FRAME MEAT THEREOF}

The present invention relates to the production of a feed additive that improves the palatability of pet food and pet food using tuna frame meat and provides antioxidant properties. More specifically, it relates to producing a protein hydrolyzed extract of tuna frame meat, Accordingly, the Maillard reaction is induced through the process of adding and heating lactic acid bacteria fermentation and reducing sugar, ultimately improving the flavor and antioxidant capacity through lactic acid bacteria fermentation and adding reducing sugar, thereby improving the palatability and antioxidant properties of pet food and snacks (pet food). It relates to a method of manufacturing a pet food additive that promotes

Tuna is a fish widely consumed around the world due to its popularity among the public. Domestic tuna production is approximately 280,000 tons as of 2021, most of which is used as raw materials for canning (agricultural and food export information, tuna production trends). When processing tuna, a large amount of processing by-products, including heads, by-products, and frames, are generated. These fishery by-products cost a lot to process, and if simply disposed of, they lead to illegal landfilling and ocean dumping in addition to wastewater leaks and bad odors, causing environmental pollution. Therefore, in order to solve problems such as environmental pollution and disposal costs, it is necessary to minimize discarded fishery by-products and find ways to utilize them to improve added value (Kim, D. Y. & Lee, J. S. (2015). Directions for Eco-friendly Utilization and Industrialization of Fishery By-products. Journal of Fisheries and Marine Science Education, 27(2), 566-575.).

Among tuna parts, frame meat, which is generated during the processing of fillets, contains some of the lean meat centered around the spine. Previous studies have reported that tuna hydrolysates promote the growth of lactic acid bacteria as a nitrogen source (Safari, R., Motamedzadegan, A., Ovissipour, M. et al. (2012). Use of Hydrolysates from Yellowfin Tuna ( Thunnus albacares) Heads as a Complex Nitrogen Source for Food Bioprocess Technol 5, 73-79. Therefore, the present invention is a feed for the purpose of improving palatability and functionality, including antioxidant ability, by inoculating the hydrolyzate obtained by enzymatic hydrolysis of tuna frame meat with lactic acid bacteria, fermenting it, and inducing the Maillard reaction through the addition of reducing sugar. A plan to utilize additives was devised.

Tuna contains flavor amino acids such as glutamic acid and aspartic acid, which are involved in sweetness and umami, so there are research results on hydrolyzing boiled liquid, one of the tuna by-products, and using the hydrolyzate to create a functional seasoning sauce (Oh. H. S. , Kim, J. S., Heu, M. S. (2007), Preparation of Functional Seasoning Sauce from Skipjack Tuna Cooking Drip. Journal of the Korean Society of Food Science and Nutrition, 36(6), 766-772. Research and cases using framed meat generated during processing as feed and food additives are limited.

Previous research and inventions using tuna by-products include the development of food materials and manufacturing of health foods using tuna bone meal (Domestic Patent Registration No. 1020050022235) and food additives such as the manufacturing method of tuna natural flavor using tuna by-products (Domestic Patent Registration No. 1020120144704). Since it is limited to the use of tuna by-products from food processing, its use as a feed additive for pets is limited. In addition, cases of using fish by-products as livestock feed materials, such as solid fish meat using fish by-products and feed containing the same (domestic patent registration no. 1020060081855) and fish meat extract using fish by-products and livestock feed containing the same (domestic patent registration no. 1020000047040). However, there are no cases of improving palatability and functional properties of marine by-products by adding lactic acid bacteria for the fermentation process after hydrolysis.

Lactic acid bacteria used as starter strains have a significant impact on flavor by rapidly lowering pH by producing lactic acid and producing various organic acids, and fermentation by lactic acid bacteria can improve nutritional value, palatability, and antibacterial properties (Benno K. 2003. Production and microbiological characteristics of fermented sausages. 23.4: 361-375; Jovanovic M. et al. Functionality and palatability of yogurt produced using beetroot pomace flour granulated with lactic acid bacteria. 1696.). In addition, a previous study showed that yellowfin tuna head hydrolyzate was a nitrogen source for the growth of lactic acid bacteria, and that microbial media containing yellowfin tuna head hydrolyzate showed a higher growth rate of lactic acid bacteria compared to commercial media. This means that the hydrolyzate of tuna by-products can be usefully used as a complex nitrogen source for lactic acid bacteria, so the antioxidant ability of the hydrolyzate can be expected due to the growth of lactic acid bacteria (Safari R. 2012. Use of hydrolysates from yellowfin tuna (Thunnus albacares) heads as a complex nitrogen source for lactic acid bacteria 5.1: 73-79.).

In other words, tuna by-products were hydrolyzed to use them as food ingredients, but in the manufacturing method of feed additives to improve the palatability of companion animals, lactic acid bacteria fermentation of the hydrolyzate is performed to increase flavor and nutritional value as well as antioxidant properties. , it is possible to develop feed additive processing technology with improved palatability through the creation of Maillard reactants that provide umami and savory taste.

(Non-patent Document 1) Kim, D. Y. & Lee, J. S. (2015). Directions for Eco-friendly Utilization and Industrialization of Fishery By-products. Journal of Fisheries and Marine Science Education, 27(2), 566-575. (Non-patent Document 2) Safari, R., Motamedzadegan, A., Ovissipour, M. et al. (2012). Use of Hydrolysates from Yellowfin Tuna (Thunnus albacares) Heads as a Complex Nitrogen Source for Lactic Acid Bacteria. Food Bioprocess Technol 5, 73-79. (Non-patent Document 3) Oh. H. S., Kim. J. S., Heu. M. S. (2007). Preparation of Functional Seasoning Sauce Using Enzymatic Hydrolysates from Skipjack Tuna Cooking Drip. Journal of the Korean Society of Food Science and Nutrition, 36(6), 766-772. (Non-patent Document 4) Benno K. 2003. Production and microbiological characteristics of fermented sausages. Food Science of Animal Resources. 23.4: 361-375; Jovanovic M. et al. 2021. Functionality and palatability of yogurt produced using beetroot pomace flour granulated with lactic acid bacteria. Foods. 10.8: 1696. Domestic registered patent No. 10-2005-0022235 Domestic registered patent No. 10-2012-0144704 Domestic Registered Patent No. 10-2006-0081855 Domestic registered patent No. 10-2000-0047040

The present invention was created to solve the above problems, and the purpose of the present invention is to improve palatability and functional properties by hydrolyzing tuna frame meat, fermenting lactic acid bacteria, and adding reducing sugars to palatability enhancers and foods containing the nutritional characteristics of tuna. The purpose is to provide improved, high-quality pet food additives.

The technical problems to be solved by the invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below. You will be able to.

The method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat to improve the antioxidant ability of pet food according to the present invention is,

A washing step of washing tuna by-products;

A protein hydrolysis step of preparing a tuna hydrolyzate by mixing a proteolytic enzyme with the washed tuna by-product;

An enzyme inactivation step of heating the tuna hydrolyzate to inactivate the proteolytic enzyme;

A filtration step of filtering the tuna hydrolyzate in which the proteolytic enzyme is inactivated;

A lactic acid bacteria fermentation step of producing a fermented tuna product by inoculating the filtered tuna hydrolyzate with a mixed strain of lactic acid bacteria;

A Maillard reaction step of adding reducing sugar to the tuna fermentation and heating it to produce a tuna Maillard reaction product;

A powdering step of concentrating and powdering the tuna Maillard reaction product.

As a means of solving the above problem, the present invention provides a feed additive manufactured by hydrolyzing tuna frame meat, lactic acid bacteria fermentation, and Maillard reaction, and when added to feed and pet food production, palatability and antioxidant capacity are improved. It is effective.

Figure 1 is a flow chart of the manufacturing method of the lactic acid bacteria fermented feed additive derived from tuna frame meat to improve the antioxidant ability of pet food according to the present invention.
Figure 2 is a graph confirming the browning index of the Maillard reaction product derived from tuna by-product prepared in Example 3, according to an embodiment of the present invention. (Control, unfermented tuna frame meat hydrolyzate; Starter culture A, tuna frame meat hydrolyzate with starter culture A; Starter culture B, tuna frame meat hydrolyzate with starter culture B. All values are mean ± standard error. ah Bar graph Different initials are recognized as significant (p<0.05))
Figure 3 is a graph confirming the results of protein electrophoresis of the Maillard reaction product derived from tuna by-product prepared in Example 3, according to an embodiment of the present invention. (a) is tuna frame meat hydrolyzate, (b) is frame meat hydrolyzate with starter culture A, and (c) is frame meat hydrolyzate with starter culture B. (PM: Protein standard marker, Raw Control: raw protein extract, Raw Hydrolysate: raw hydrolyzate, No sugar: simple heating treatment, Xylose: 1% (w/v) xylose addition treatment, Glucose: 1% (w/ v) Glucose addition treatment group, Xylose+Glucose: 0.5% (w/v) xylose + 0.5% (w/v) glucose addition treatment group)
Figure 4 shows the results of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity of the Maillard reaction product derived from tuna by-product prepared in Example 1, according to an embodiment of the present invention. It's a graph. (Control, unfermented tuna frame meat hydrolyzate; Starter culture A, tuna frame meat hydrolyzate with starter culture A; Starter culture B, tuna frame meat hydrolyzate with starter culture B)
Figure 5 is a graph confirming the reducing power results of the Maillard reaction product derived from tuna by-product prepared in Example 1, according to an embodiment of the present invention. (Control, unfermented tuna frame meat hydrolyzate; Starter culture A, tuna frame meat hydrolyzate with starter culture A; Starter culture B, tuna frame meat hydrolyzate with starter culture B)
Figure 6 is a graph showing the results of multivariate analysis of the flavor component pattern of hydrolyzate derived from tuna frame meat, according to an embodiment of the present invention. (A, control group of tuna frame hydrolyzate with starter culture A; B, xylose Maillard reaction treatment group of tuna frame meat hydrolyzate with starter culture A added; C, glucose Maillard reaction treatment group of tuna frame hydrolyzate with starter culture A added D, xylose + glucose Maillard reaction treatment of tuna frame meat hydrolyzate with starter culture A; F, non-fermented tuna frame hydrolyzate; , xylose Maillard reaction treatment of non-fermented added tuna frame meat hydrolyzate; I, xylose + glucose Maillard reaction treatment of non-fermented tuna frame meat hydrolyzate; Treatment group; J, simple heating treatment of unfermented tuna frame meat hydrolyzate; L, xylose Maillard reaction treatment of tuna frame meat hydrolyzate with starter culture B; N, glucose Maillard reaction treatment of tuna frame meat hydrolyzate with starter culture B, O, simple tuna frame hydrolyzate with starter culture B; heating treatment area)

The terms used in this specification will be briefly explained, and the present invention will be described in detail.

The terms used in the present invention are general terms that are currently widely used as much as possible while considering the functions in the present invention, but this may vary depending on the intention or precedent of a person working in the art, the emergence of new technology, etc. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.

When it is said that a part “includes” a certain element throughout the specification, this means that it does not exclude other elements, but may further include other elements, unless specifically stated to the contrary.

Below, with reference to the attached drawings, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

Specific details, including the problem to be solved by the present invention, the means for solving the problem, and the effect of the invention, are included in the examples and drawings described below. The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings.

Hereinafter, the present invention will be described in more detail with reference to the attached drawings.

As shown in Figure 1, the method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat to improve the antioxidant capacity of pet food according to the present invention is carried out by the following method.

First, the first step (S10) is a washing step. In the first step (S10), the product is washed with distilled water at least three times and then cut into pieces of 1.0 to 2.0 kg. It is preferable to cut under the above conditions so that enzymatic hydrolysis can be easily performed in the second step (S20) below.

Next, the second step (S20) is the protein hydrolysis step.

More specifically, in the second step (S20), Alcalase, a protease, is added to a buffer solution of 1.3 to 1.7 L of distilled water at pH 8.0 at a concentration of 2.0 to 2.5 AU/kg. Thereafter, the tuna by-products are mixed together and stirred with a propeller at 50 to 60° C. for 50 to 70 minutes.

The tuna by-product is hydrolyzed by preparing an equal amount of the buffer solution by weight.

The protein hydrolysis is preferably performed by stirring at 50 to 60° C. for 1 hour. More preferably, it is performed at 55°C for 1 hour. If the protein hydrolysis is performed below 50°C, there is a risk that the hydrolysis may not be carried out well, and if the protein hydrolysis is performed above 60°C, there is a risk that the proteolytic enzyme may be deteriorated. Therefore, it is preferable to perform the protein hydrolysis under the above conditions. In addition, if the protein hydrolysis is performed for less than 1 hour, there is a risk that the hydrolysis may not be carried out well, and if the protein hydrolysis is performed for more than 1 hour, there is a risk that the proteolytic enzyme may be deteriorated, so it is preferable to perform it under the above conditions. do.

Next, the third step (S30) is the enzyme inactivation step. In the third step (S30), the tuna hydrolyzate is heated to inactivate the proteolytic enzyme.

More specifically, in the third step (S30), the mixture is heated at 80 to 90° C. for 10 to 30 minutes and then cooled to room temperature.

The enzyme inactivation step is preferably performed by heating at 80 to 90° C. for 20 minutes and then cooling to room temperature. More preferably, it is heated at 85°C for 20 minutes and then cooled to room temperature. If the enzyme inactivation is performed below 80 ℃, there is a risk that the enzyme inactivation will not occur easily, and if the enzyme inactivation is performed above 90 ℃, there is a risk that the intestines of the tuna will deteriorate, so it is performed under the above conditions. It is desirable to do so. If the enzyme inactivation is performed for less than 20 minutes, there is a risk that the enzyme inactivation will not occur easily, and if the enzyme inactivation is performed for more than 20 minutes, there is a risk that the intestines of the tuna will deteriorate, so it is performed under the above conditions. It is desirable to do so.

Next, the fourth step (S40) is a filtration step. In the fourth step (S40), the tuna hydrolyzate in which the proteolytic enzyme has been inactivated is filtered.

More specifically, in the fourth step (S40), filtration is performed using a sieve with a size of 500 ㎛/35 and a line thickness of 315 ㎛.

Next, the fifth step (S50) is the lactic acid bacteria fermentation step. In the fifth step (S50), fermented tuna is produced by inoculating the filtered tuna hydrolyzate with a mixed strain of lactic acid bacteria.

More specifically, in the fifth step (S50), 0.5 to 1 part by weight of mixed strain B is mixed with 1 part by weight of mixed strain A and fermented at 35 to 45 ° C. for 11 to 13 hours.

The mixed strain A is a mixed strain of Bifidobacterium lactis, Lactobacillus acidophilus, and Streptococcus thermophilus, and the mixed strain B is Lactobacillus bulgaricus. delbrueckii subsp. bulgaricus) and Streptococcus thermophilus.

Next, the sixth step (S60) is the Maillard reaction step. In the sixth step (S60), reducing sugar is added to the tuna fermentation and heated to prepare a tuna Maillard reaction product.

More specifically, the sixth step (S60) is performed by adding at least one of xylose or glucose as the reducing sugar and heating at 90 to 100 ° C. for 50 to 70 minutes. Most preferably, the Maillard reaction is induced by adding a mixture of 0.5% (w/v) glucose and 0.5% (w/v) xylose, or 1.0% (w/v) glucose or 1.0% (w/v) ) Xylose is added to induce the Maillard reaction.

The Maillard reaction step is performed by heating at 90 to 100° C. for 1 hour. If the Maillard reaction is performed at less than 90°C, there is a risk that the Maillard reaction may not be performed well, and if it is performed above 100°C, there is a risk that the antioxidant activity may be minimal due to the minimal interaction due to the reducing sugar pretreatment. In addition, if the Maillard reaction is performed for less than 1 hour, there is a risk that the flavor of the Maillard reaction produced will be insignificant, and if the Maillard reaction is performed for more than 1 hour, there is a risk that the volatile flavor of the Maillard reaction produced may be deteriorated, making it a risk for pets. Since there is a risk that the palatability of the food may be lowered, it is preferable to perform under the above conditions.

Next, the seventh step (S70) is the powdering step. In the seventh step (S70), the Maillard reaction product produced through the Maillard reaction is cooled, freeze-dried, and powdered.

Hereinafter, comparative examples and examples prepared by conventional methods will be compared, and the present invention will be described in more detail through experimental examples. The purpose, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced here are provided to enable the idea of the present invention to be sufficiently conveyed to those skilled in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

Example 1. Preparation of tuna hydrolyzate

Frozen tuna by-products were sufficiently thawed at refrigerated temperature for 24 hours before use. Thawed tuna by-products were selected and used to prepare hydrolysates. Tuna by-products were thoroughly washed at least three times in running tap water and then cut into appropriate sizes. A shredder, etc. can be used for industrial productivity. Proteinase (Alcalase) was added to 1.5 kg of tuna by-products at a concentration of 2.4 AU/kg in 1.5 L of a pH 8.0 solution, and enzymatic hydrolysis was performed. Protein hydrolysis was performed at 55 °C for 1 hour by stirring with a propeller, and hydrolysis was stopped by heating at 85 °C for 20 minutes to inactivate the enzyme. The protein hydrolyzate derived from tuna by-products was cooled at room temperature and filtered using a test sieve (size 500 ㎛/35, line thickness 315 ㎛, 885705, CHUNG GYE SANG GONG SA, Seoul, Korea). The filtered protein hydrolyzate derived from tuna by-products was refrigerated at 4 °C until used in experiments.

Example 2. Tuna hydrolyzate lactic acid bacteria inoculation

In this experiment, tuna by-products were enzymatically hydrolyzed and then mixed with lactic acid bacteria starter culture A (Bifidobacterium lactis, Lactobacillus acidophilus, and Streptococcus thermophilus mixed strain, 0.056 mg/mL) and starter culture B (Lactobacillus delbrueckii subsp. bulgaricus) to enhance antioxidant properties. Fermentation was performed by inoculating Streptococcus thermophilus mixed strains (0.04 mg/mL). The lactic acid bacteria fermentation treatment group, excluding the non-fermentation control group, was fermented at 40 °C for 12 hours.

Experimental Example 1. Evaluation of tuna protein hydrolysis characteristics

The hydrolysis characteristics of the tuna-derived protein hydrolysates obtained in Examples 1 and 2 were evaluated in terms of degree of hydrolysis as follows.

The degree of hydrolysis of tuna by-products according to the type of lactic acid bacteria was measured using the O-phtahldialdehyde (OPA) method (Aspevik T, Egede-Nissen H, Oterhals L., 2016, A Systematic Approach to the Comparison of Cost Efficiency of Endopeptidases for the Hydrolysis of Atlantic Salmon (Salmo salar) By-Products. Biotechnol. 3 mL of OPA reagent was added to 400 μL of tuna hydrolyzate, reacted for 2 minutes, and absorbance was measured at 340 nm. A standard curve was obtained from the absorbance measured by diluting leucine (L-leucine) to a concentration of 0 to 2000 μg/mL of OPA leucine. The degree of protein hydrolysis for each type of tuna by-product starter culture was calculated by calculating the leucine concentration according to the absorbance of the sample from the OPA leucine standard curve.

item Non-fermented
tuna frame meat
hydrolyzate Starter Culture A Fermented Tuna Frame Meat Hydrolyzate Starter Culture B Fermented Tuna Frame Meat Hydrolyzate Protein hydrolysis degree (μg/mL) 37.40± ^032b 39.77± ^0.31a 37.37± ^0.26b All values are mean ± standard error.
^a,b Different initials in the same row are recognized as significant (p<0.05).

As a result, as shown in Table 1, the degree of protein hydrolysis of the tuna hydrolyzate was higher in the hydrolyzate with Starter Culture A compared to the unfermented tuna hydrolyzate and the hydrolyzate with Starter Culture B (p<0.05).

Experimental Example 2. Free amino acid analysis

The free amino acid content of the hydrolyzate of lactic acid bacteria-inoculated tuna frame meat obtained in Example 2 was evaluated as follows.

(1) Free amino acid analysis for each lactic acid bacteria fermentation treatment of tuna frame meat was analyzed after pretreatment using the trichloroacetate method (Ministry of Food and Drug Safety, Food and Food Additives Code). An equal volume of 16% trichloroacetic acid solution was added to 3 mL of hydrolyzate of lactic acid bacteria-inoculated tuna frame meat and heated in a constant temperature water bath preheated to 100 °C for 15 minutes. After cooling for 30 minutes, the supernatant obtained after centrifugation at 3000 rpm for 15 minutes was filtered using a 0.45 ㎛ syringe filter, diluted 10 times, and used for free amino acid analysis. Free amino acid analysis was performed using an Amino Acid Analyzer (II) (Hitachi (L-8900)).

Item (mg/mL) Non-fermented
tuna frame meat
hydrolyzate Starter Culture A Fermented Tuna Frame Meat Hydrolyzate Starter Culture B Fermented Tuna Frame Meat Hydrolyzate essential amino acids Phe 885.9±55.3 811.4±75.5 763.7±144.0 Lys 579.0± ^6.1a 188.4±15.4 ^c 240.8± ^1.0b Met 681.0±50.6 637.1±2.4 715.0±4.5 Thr 275.7±18.5 188.8±25.2 192.2±29.3 Val 424.6± ^30.2ab 349.3± ^45.6b 503.0± ^3.2a Ile 573.4±16.4 439.2±29.5 487.8±71.2 Leu 1474.0± ^0.5a 1036.0±108.2 ^b 903.0±78.8 ^b non-essential amino acids Asp 210.0± ^2.9c 521.9± ^6.8a 262.2±7.5 ^b Tau 480.5±32.6 458.2±15.2 493.5±49.8 Ser 142.4± ^0.9c 223.4± ^13.6a 190.3±3.1 ^b Glu 527.8± ^11.2a 475.2± ^55.6ab 381.6±16.8 ^b Gly 158.1±3.2 232.7±144.5 211.3±1.5 Ala 388.5±1.9 730.2±199.8 747.6±22.2 Tyr 582.7±9.3 ^b 851.2.± ^68.6a 516.9±83.3 ^b Orn 244.2±6.4 ^b 92.3± ^0.7c 326.7± ^28.6a His 1093.6±38.0 1035.3±29.2 1046.0±86.3 Ans 5846.1±288.6 4835.3±170.7 4938.9±725.0 Car 170.2±170.2 115.1±115.1 188.5±188.5 Arg 302.2±31.4 ^c 1287.3± ^17.1a 669.9±24.6 ^b Hypro - ^(One) - - Pro - 25.4± ^25.4a - All values are mean ± standard error.
^ac Different initials in the same row are recognized as significant (p<0.05).
¹⁾ Below the minimum detection limit

As a result, as shown in Table 2, seven types of essential amino acids were detected in the hydrolyzate of tuna frame meat, including phenylalanine, lysine, methionine, tyrosine, valine, isoleucine, and leucine. Lactic acid fermentation had a significant effect on the essential amino acid content of tuna frame meat hydrolyzate (p<0.05). Valine showed significantly higher levels in the Starter Culture A B fermentation treatment group, and lysine and leucine showed significantly higher levels in the non-fermentation treatment group (p<0.05). There was no significant difference in methionine, phenylalanine, methionine, threonine, and isoleucine between fermentation treatments (p<0.05).

In the case of non-essential amino acids, aspartic acid, serine, tyrosine, and arginine showed significantly higher levels in the Starter Culture A fermentation treatment group, and ornithine showed higher levels in the Starter Culture B fermentation treatment group (p<0.05). Glutamic acid showed high levels in the non-fermented group, and there was no significant difference in taurine, glycine, alanine, histidine, anserine, and carnosine between the fermented groups (p<0.05). Hydroxyproline was measured below the minimum detection limit, and proline was detected only in the Starter Culture A treatment group (p<0.05).

Example 3. Maillard reaction of tuna by-product hydrolyzate

In this experiment, by-products from each part of tuna were enzymatically hydrolyzed, and then Maillard reaction was performed by adding reducing sugars xylose and glucose to improve the palatability and antioxidant properties of each part. Excluding the control group, simple heat treatment, 1% (w/v) xylose treatment, 1% (w/v) glucose treatment, and 0.5% (w/v) xylose + 0.5% (w/v) glucose treatment were each After adding the reducing sugar required for the concentration, it was heated at 95 °C for 1 hour to obtain a Maillard reaction. The Maillard reactant was cooled, freeze-dried, and the powdered Maillard reactant was used in the analysis experiment.

Experimental Example 3. Evaluation of the degree of Maillard reaction

The production characteristics of the Maillard reaction product derived from the tuna by-product prepared in Example 3 were evaluated by browning index and protein electrophoresis (protein SDS-PAGE) as follows.

(1) The degree of browning of the Maillard reaction product derived from tuna by-products was compared by diluting the sample 10 times with distilled water, centrifuging it at 3,000 × g for 10 minutes, and measuring the absorbance of the supernatant at 420 nm.

As a result, as shown in FIG. 2, an interaction according to the Maillard reaction conditions was shown (p<0.05), and the treatment group in which only the Maillard reaction was performed without lactic acid bacteria fermentation showed a high degree of browning (p<0.05). This is believed to be the result of lactic acid bacteria utilizing some free amino acids as a nitrogen source during the fermentation process. In addition, compared to simple heating treatment, a higher degree of browning could be expected when heating with the addition of reducing sugars such as xylose or glucose (p<0.05). In particular, the highest degree of browning was observed when the Maillard reaction was induced by adding a mixture of 0.5% (w/v) glucose and 0.5% (w/v) xylose.

(2) Protein electrophoresis was performed by applying the method of Laemmli (1970). All treatments were diluted so that the protein concentration was 5 mg/mL. The sample was diluted with buffer (312.5 mM Tris-HCl (pH 6.8), 50% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.05% Bromophenol blue) at a 4:1 ratio and placed in a constant temperature water bath preheated to 100 °C. After heat denaturation for 5 minutes, electrophoresis (mini protein tetra cell, Bio-Rad, CA, USA) was performed. 20 uL of sample was injected into 18% polyacrylamide gel (4% stacking gel, 18% separating gel). The injected protein was passed through a stacking gel at a voltage of 70 V for about 10 minutes, and then passed through a separating gel at 100 V. After electrophoresis was completed, the gel was treated with a staining solution (0.25% (w/v) Coomassie blue R-250, 50% (v/v) methanol, 40% (v/v) distilled water, 10% (v/v) acetic acid. ) and then destained with a de-staining solution (50% (v/v) methanol, 40% (v/v) distilled water, 10% (v/v) acetic acid). To confirm the molecular weight, a standard material (Dokdo-mark EBM-1032, elpis, Deajeon, Korea) was used.

As a result, as shown in Figure 3, a protein band presumed to be derived from myofibrillar and connective tissue proteins was observed in the untreated raw material, and protein hydrolysis also progressed through the disappearance of the corresponding band and the appearance of a low-molecular-weight band after protein hydrolysis. Confirmed. Furthermore, compared to the simple heating treatment, the appearance of additional polymer bands and an increase in intensity were observed in the treatment with reducing sugar added. This is believed to be due to the formation of glycoprotein polymers following the Maillard reaction.

Experimental Example 4. Evaluation of antioxidant activity of Maillard reaction product derived from tuna by-products

In Example 1, the antioxidant activity of the Maillard reaction product derived from tuna by-products was evaluated in terms of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging ability and reducing power as follows.

(1) ABTS (2, 2'-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging ability is obtained by mixing 7.4mM ABTS and 2.6mM potassium persulphate in a 1:1 ratio and reacting for 12 hours in the dark at room temperature. ABTS reagent was prepared. Afterwards, the prepared ABTS reagent was mixed with methanol at a ratio of 1:15 and used to measure ABTS radical scavenging ability. 2.85 mL of ABTS solution was mixed with 0.15 mL of the sample, reacted for 2 hours in the dark at room temperature, and then the absorbance was measured at 734 nm. Trolox was used as a standard material, and a standard line was obtained from a standard material at a concentration of 50-600 μM and calculated by substituting the measured absorbance value of the sample. ABTS radical scavenging activity was expressed as μmol Trolox equivalent (TE)/mL.

(2) Reducing power is obtained by adding 2 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2 mL of 1% potassium ferricyanide to 2 mL of sample, reacting at 50 °C for 20 minutes, and then adding 2 mL of 10% trichloroacetic acid. After addition, centrifugation was performed at 1500 × g for 20 minutes. Afterwards, 2 mL of the supernatant was transferred to a test tube, mixed with 2 mL of distilled water and 0.4 mL of 0.1% ferric chloride, reacted for 10 minutes, and the absorbance was measured at 700 nm.

As a result, as shown in FIG. 4, there was an interaction between lactic acid bacteria inoculation and Maillard reaction conditions (p<0.05), and the lactic acid bacteria fermentation treatment group showed significantly higher ABTS radical cation scavenging ability (p<0.05). In particular, when treated with 1% (w/v) glucose under Maillard reaction conditions, high ABTS radical cation scavenging ability could be expected in the treatment group inoculated with lactic acid bacteria. In terms of reducing power characteristics, a clear improvement in reducing power was observed when inoculating lactic acid bacteria and adding xylose to induce the Maillard reaction (p<0.05).

Experimental Example 5. Flavor pattern analysis of hydrolyzate derived from tuna frame meat

(1) Analysis of volatile aroma components using electronic nose

An electronic nose system (HERACLES Neo, Alpha MOS) was used to analyze volatile aroma components present in the sample. The electronic nose's analysis column was MXT-5 column (Alpha MOS). 5 g of sample was dissolved in 100 mL of purified water, filtered through filter paper (Whatman No. 1), and 4 mL was taken and used for analysis. It was placed in a headspace vial (22.5 × 75 mm, PTEE/silicone septum, aluminum cap) for electronic nose analysis and stirred at 500 rpm at 50 °C for 20 minutes to saturate the inside of the vial with volatile aroma components. Volatile scent components were collected through an automatic sample collector attached to the electronic nose, and 2,000 μL of the collected volatile scent components were taken using a syrige and then injected into the gas chromatography injection port mounted on the electronic nose. As for the analysis conditions, the hydrogen gas flow rate was set to 1 mL/min, the acquisition time was 227 seconds, the trap absorption temperature was 40 °C, and the trap desorption temperature was 250 °C. The oven temperature was maintained at 40 °C for 5 seconds, then increased to 270 °C at a rate of 4 °C/s and held for 30 seconds. The retention index based on carbon number was based on Kovat’s index library, and the separated peak components were identified using AroChemBase (Alpha MOS) included in the electronic nose. Electronic nose analysis was performed a total of three times per sample, and the odor pattern was confirmed using multivariate analysis.

As a result, as shown in FIG. 6, the volatile flavor pattern of the Maillard reaction product derived from tuna frame meat was measured with an electronic nose and principal component analysis was performed, and the total variation was 89.661%. PC1 on the X axis showed an explanatory power of 60.688% and PC2 on the Y axis showed an explanatory power of 28.973%. Some treatments (A, C, E) of hydrolyzate of tuna frame meat with starter culture A and some treatments (J, H, F) of hydrolyzate of non-fermented tuna frame meat were located in the positive region for PC1, while all treatments with addition of starter culture B Tuna frame meat hydrolyzate treatment groups (K ~ O) formed clusters in the negative area. This means that the flavor pattern of tuna frame meat changes significantly due to the addition of lactic acid bacteria and fermentation. However, xylose (B, G, L) and xylose+glucose added Maillard reaction treatments (D, I, N) formed clusters in the negative area of PC1 and the positive area of PC2, resulting in the addition of reducing sugar and the Maillard reaction. The flavor pattern of the tuna frame meat hydrolyzate showed a tendency to become similar.

As a means of solving the above problem, the present invention provides a feed additive manufactured by hydrolyzing tuna frame meat, lactic acid bacteria fermentation, and Maillard reaction, and when added to feed and pet food production, palatability and antioxidant capacity are improved. It is effective.

As such, a person skilled in the art will understand that the technical configuration of the present invention described above can be implemented in other specific forms without changing the technical idea or essential features of the present invention.

Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive, and the scope of the present invention is indicated by the claims described later rather than the detailed description above, and the meaning and scope of the claims and their equivalents. All changes or modified forms derived from the concept should be construed as falling within the scope of the present invention.

S10. washing steps
S20. Protein hydrolysis step
S30. Enzyme inactivation step
S40. filtration stage
S50. Lactic acid bacteria fermentation stage
S60. Maillard reaction steps
S70. powdering step

Claims (12)

A washing step of washing tuna by-products;
A protein hydrolysis step of preparing a tuna hydrolyzate by mixing a proteolytic enzyme with the washed tuna by-product;
An enzyme inactivation step of heating the tuna hydrolyzate to inactivate the proteolytic enzyme;
A filtration step of filtering the tuna hydrolyzate in which the proteolytic enzyme is inactivated;
A lactic acid bacteria fermentation step of producing a fermented tuna product by inoculating the filtered tuna hydrolyzate with a mixed strain of lactic acid bacteria;
A Maillard reaction step of adding reducing sugar to the tuna fermentation and heating it to produce a tuna Maillard reaction product;
A method for producing a lactic acid bacteria-fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, comprising a powdering step of concentrating and powdering the tuna Maillard reaction product.
According to clause 1,
In the protein hydrolysis step,
The proteolytic enzyme is alcalase,
A lactic acid bacteria fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized in that hydrolysis is performed by mixing the by-product of the washed tuna with a buffer solution containing the alcalase. Manufacturing method.
According to clause 2,
A method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, wherein the buffer solution has a pH of 8.0.
According to clause 2,
A method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized in that the proteolytic enzyme is mixed at a concentration of 2.0 to 2.5 AU/kg.
According to clause 1,
In the protein hydrolysis step,
A method for producing a lactic acid bacteria-fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized in that it is carried out by stirring at 50 to 60 ° C. for 50 to 70 minutes.
According to clause 1,
The enzyme inactivation step is,
A method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized by heating at 80 to 90 ° C. for 10 to 30 minutes and then cooling at room temperature.
According to clause 1,
In the lactic acid bacteria fermentation step,
Method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat to improve the antioxidant capacity of pet food, characterized by inoculating and fermenting the mixed strain A and mixed strain B below:
Mixed strain A: Mixed strain of Bifidobacterium lactis, Lactobacillus acidophilus, and Streptococcus thermophilus
Mixed strain B: Mixed strain of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.
According to clause 7,
A method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized in that 0.5 to 1 part by weight of mixed strain B is mixed and fermented with respect to 1 part by weight of mixed strain A.
According to clause 1,
The lactic acid bacteria fermentation step is,
A method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized by fermentation at 35 to 45 ° C. for 11 to 13 hours.
According to clause 1,
The Maillard reaction step is,
A method for producing a lactic acid bacteria fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized in that adding at least one of xylose or glucose as the reducing sugar.
According to clause 1,
The Maillard reaction step is,
A method for producing a lactic acid bacteria-fermented feed additive derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized in that it is performed by heating at 90 to 100 ° C. for 50 to 70 minutes.
A feed additive fermented with lactic acid bacteria derived from tuna frame meat for improving the antioxidant capacity of pet food, characterized in that it is manufactured by the method of claims 1 to 11.

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