Process and product of fermented acorns using baker's yeast for fish feeding

Abstract

The search for new food raw materials through various processing methods is of great importance in food security and the economy of society. More than 5 million hectares of Iran are covered with oak forests. In addition to having nutritional compounds, oak fruit contains a bitter and anti-nutritional substance called tannin. Anti-nutritional factors can be greatly reduced by various methods, including a combination of soaking (physical) and fermentation (biological). In this invention, through a new process and in order including: Soaking the acorn kernels without the outer and inner shells in plain water with a weight-volume ratio of 1 to 5, placing the soaked acorn kernels in an incubator at a temperature of 50 degrees Celsius for 24 hours and changing the water every 8 hours, washing the acorn kernels with plain water, grinding the acorn kernels, dissolving 10 grams of instant dry baker's yeast per 100 grams of acorns in 50 milliliters of lukewarm water, adding the yeast solution to the ground acorn kernels, fermenting the acorns in an incubator for 24 hours at a temperature of 30 degrees Celsius, inactivating the fruits By fermenting acorns for 10 minutes at 70 degrees Celsius, drying the acorns at 60 degrees Celsius, and then grinding them, acorn flour with high digestibility can be produced for use in the diet of carp, which is one of the most important species of farmed fish in Iran and the world. In intensive aquaculture, feed sometimes accounts for up to 80 percent of the current costs, and using the results of this invention will lead to increased food security and improved economic conditions for the community.

Description

Dear Applicant, the description of the invention should be presented in the following 9 paragraphs and in the same order. For Persian typing, use the B Nazanin font with a font size of 12 and for English typing, use the Arial font with a font size of 10. Description of the invention Title of the invention (as stated in the declaration) Process and product of fermented acorns using baker's yeast for fish feeding Technical background of the relevant invention "This invention is related to the field of aquatic feed manufacturing industry and for the first time, attempts have been made to produce oak flour fermented by baker's yeast in accordance with the digestive ability of carp." Technical problem and stating the objectives of the invention The lack of diversity of raw materials is a major challenge for the aquatic feed industry. More than 5 million hectares of Iran is covered by oak forests, but the valuable fruit of this native tree is not used in the production of aquatic feed. In addition to containing nutritious compounds such as carbohydrates, vitamins, minerals, etc., oak fruit is a substance rich in tannins. Tannins are the cause of the bitter taste of oak and, in higher concentrations, have anti-nutritional properties. Ingestion of large amounts of tannins may cause damage to the epithelial tissues of the intestine, liver, and kidney. Anti-nutritional factors can be reduced through various processing methods, including soaking and fermentation under specific conditions. The purpose of this invention is to increase the possibility of using oak fruit, increase the diversity of raw materials for the production of aquatic feed, reduce the price of fish feed due to the lower price of oak compared to a large number of plant food raw materials, and increase the food security of the community. A description of the state of the prior art and the history of developments related to the claimed invention. No invention has been registered in the field of fish food production using fermented acorns so far. In the only similar study conducted on carp by Naroui and Alamdari in 1401, published in the Iranian Journal of Fisheries, the use of fermented acorns did not have a significant negative effect on growth performance, survival rate, feed conversion ratio, feed intake rate, and protein and fat yield rates, but it caused a significant decrease in protein and fat reserves, as well as a decrease in carcass quality compared to the control treatment. In the study by Naroui and Alamdari in 1401, dry active baker's yeast (instant) was used at a dose of 1.5 grams per 100 grams of acorns in the diet of carp fish. However, in this invention, six different doses of yeast were studied in 12 treatments based on chemical analysis (protein, fat, and phenolic compounds) and digestibility (carbohydrates and protein) of acorns, and the decisive superiority of the dose of dry active baker's yeast (instant) equal to 10 grams per 100 grams of acorns was proven. It should be noted that an invention is patentable if it contains a new invention and has industrial application. A new invention is something that did not exist in the prior art or industry and is not known or obvious to a person of ordinary skill in the art. Currently, no aqua feed factory in Iran uses acorn flour in fish feed, and therefore the existence of an inventive component in the description of this invention is obvious. If fermented acorns are used according to the specific conditions mentioned in this invention, we can see an improvement in the nutritional value of the acorns and fish feed. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention In this invention, in order to reduce the bitterness (tannin) and also increase the nutritional value and digestibility of acorns, the method of soaking and then fermenting acorns under specific conditions is used. Explain that, acorns are collected from the forest. After manually removing the fruit cup and removing its outer shell with a knife, the resulting acorn core is placed in a drying machine. After drying, the inner shell of the fruit (the acorn) is manually removed. The acorn core is soaked in ordinary water for 24 hours. Soaking the acorns is done with a weight-volume ratio of 1 to 5 in an incubator at a temperature of 50 degrees Celsius and with water changed every 8 hours. After soaking, the acorns are ground with a meat grinder. Then, 10 grams of dry active baker's yeast (instant yeast) is added to them per 100 grams of acorns and dissolved in 50 milliliters of lukewarm ordinary water. Fermentation is carried out for 24 hours in an incubator at 30°C. The fermented acorns are placed at 70°C for 10 minutes to inactivate the yeast.After inactivating the yeast, the fermented acorns are dried at 60°C. The acorns are then ground and sieved through a 250 micron sieve. This fermented acorn flour can be used in aquatic feed factories as one of the raw materials in the production of carp food. Description of the experiment This invention is the final result of an experiment conducted in the form of 13 treatments (1 control treatment and 12 fermentation treatments) according to Table 1. In this experiment, first, common carp (Cyprinus carpio) were raised for 3 weeks and fed daily until satiety, twice a day. Then, the crude enzyme was extracted from the fish intestine. 1-Enzyme extraction At the end of the rearing period, feeding was stopped for 24 hours to empty the digestive tract. Three fish with an average weight of 95.63 g and an average length of 19.5 cm were caught randomly and anesthetized by hitting the head. The entire intestine was isolated and placed on a cold Petri dish. Blood and waste were washed with cold phosphate-buffered saline (pH 19.8, 0.1 M containing 0.9% sodium chloride). The intestine was cleaned and cut into pieces with scissors, and with the help of the buffer solution, a 10% homogenate was prepared using a porcelain mortar on ice. The homogenate was centrifuged at 14,000 g for 15 minutes at 4 °C. The supernatant was stored at -60 °C until the experiment (Khan and Ghosh, 2013). The amount of protein in the enzymatic extract was measured by the Bradford method, using Coomassie Blue reagent and bovine serum albumin as a standard, based on optical absorption at 595 nm (Walker, 2002). 2- Sample preparation for digestion in vitro To 200 mg of each food sample, 40 ml of 0.1 M phosphate-buffered saline solution with a pH of 19.8 was added, followed by 1 ml of enzyme extract. The digestion reaction was carried out for 6 hours at room temperature (25°C) on a shaker at 200 rpm in the dark. Three replicates were considered for each sample. Before digestion, 1.5 ml of each mixture was removed as a control to determine protein digestibility and 1.5 ml as a control to determine carbohydrate digestibility. 3-Determination of protein digestibility A solution of 1.5 mL of undigested control or digested mixture was mixed well with 1.5 mL of cadmium ninhydrin reagent. To prepare cadmium ninhydrin reagent, 0.8 g of ninhydrin dissolved in 80 mL of 99.5% ethanol and 10 mL of acetic acid were added to 1 g of cadmium chloride dissolved in 1 mL of distilled water. The mixture was incubated for 5 min at 84°C. Then it was suddenly cooled on ice. After centrifugation at 14,000 g for 10 min, the supernatant was read at 507 nm and the concentration of active amino group was calculated using tyrosine as a standard (Kattakdad et al., 2018): Milligrams of food sample / mg of tyrosine released = In vitro protein digestibility 4-Determining the digestibility of carbohydrates A solution of 1.5 mL of undigested control or well-digested mixture with 1.5 mL of 1% dinitrosalicylic acid reagent was heated in boiling water for 5 minutes. After cooling to room temperature, the absorbance was read at 540 nm and compared with the maltose standard curve (Kattakdad et al., 2018): Milligrams of food sample / mg of maltose released = In vitro carbohydrate digestibility 5-Biochemical analysis The types of oak flour were analyzed for biochemical composition (AOAC, 2000). Briefly, crude protein was measured by the Kjeldahl method, crude fat by the Soxhlet method, ash by the furnace method, and dry matter by the oven method. Total phenolic compounds, non-tannin phenolic compounds, and condensed tannins were determined by the Folin-Sioux-Calteo method (Makkar, 2003). 6-Statistical analysis In a completely randomized design, all calculations were performed using SPSS version 20 and Excel version 2013. The normality of data distribution was controlled with the Kolmogorov-Smirnov test, the homogeneity of variance was controlled with the Levene test, the study variables were compared with the one-way analysis of variance test, and the means were compared with the Duncan test. 7- Results and Discussion As can be seen in Table 2, all experimental treatments resulted in a decrease in total phenolic compounds, non-tannin phenolic compounds, and condensed tannins compared to the control. The ash content of all treatments was significantly lower than the control, which can be explained by the initial soaking for 24 hours before fermentation. However, the importance of fat and especially protein in the nutritional value is much greater than ash. In terms of crude fat, treatment 5 did not differ significantly from other fermentation treatments, but it was significantly higher than the control treatment. After the fermentation process, the highest protein levels were found in oak of treatments 5, 6, and 12. This increase is desirable if its digestibility is also higher. According to Table 3, the protein digestibility of treatment 5 was significantly higher than that of treatments 6, 12, and the control. Based on carbohydrate digestibility, treatments 1, 2, and 5 had significantly better status compared to the control, but in aquatic animals, the nutritional importance of proteins is much higher than carbohydrates.In terms of protein digestibility, treatment number 5 was significantly superior to 1, 2, and the control. Overall, based on biochemical analysis and acorn digestibility, the superiority of treatment number 5 (baker's yeast dose equal to 10 grams per 100 grams of acorn fruit) over other treatments was proven. AOAC, 2000. Association of Official Analytical Chemists. Official Methods of Analysis 17th ed. Washington DC, 2200 pp. Kattakdad, S., Jintasataporn, O., Worawattanamateekul, W. and Chumkam, S., 2018. pH characterization of digestive enzyme and in vitro digestibility of red bee shrimp Caridina cantonensis (Decapoda: Atyidae). Journal of Aquaculture Research and Development, 9 (2): 1-6. DOI: 10.4172 / 2155-9546.1000522 Khan, A. and Ghosh, K., 2013. Phytic acid-induced inhibition of digestive protease and α-amylase in three Indian major carps: An in vitro study. Journal of the world aquaculture society, 44 (6): 853-859. Makkar, HPS, 2003. Quantification of tannins in tree and shrub foliage. A laboratory manual. FAO / IAEA. Doi:10.1007 / 978-94-017-0273-7 Walker, JM, 2002. The protein protocols handbook, second ed. Humana Press. Totowa, New Jersey, 1146 p. Explanation of shapes, maps and diagrams Table 1 - Summary of types of oak fermentation treatments using baker's yeast Soaking treatment with a weight-volume ratio of 1:5 and for 24 hours at 50°C before fermentation Yeast dosage used in grams per 100 grams of acorns During fermentation for 24 hours at 30°C 1 Soaking in ordinary water and changing the water every 8 hours 0.5 2 1.5 3 3 4 5 5 10 6 15 7 Soaking in 0.1 M sodium hydroxide solution for 8 hours and then washing the acorns and soaking in distilled water for 16 hours and changing the distilled water every 8 hours 0.5 8 1.5 9 3 10 5 11 10 12 15 13 (control) No soaking No fermentation Table 2 - Chemical analysis of different types of oak flour in terms of percentage of dry matter (standard error ± mean of three replicates) Treatment Crude protein Crude fat Total ash Phenolic compounds Non-tannin phenolic compounds Dense tannins 1 m08 / 0±80 / 2 bc35 / 0±74 / 5 g03 / 0±51 / 0 b56 / 0±07 / 5 de02 / 0±17 / 0 b00 / 0±01 / 0 2 g08 / 0±70 / 3 bc39 / 0±82 / 5 g01 / 0±57 / 0 bc15 / 0±63 / 4 de01 / 0±16 / 0 b00 / 0±01 / 0 3 f07 / 0±37 / 4 bc21 / 0±69 / 5 f01 / 0±72 / 0 b27 / 0±92 / 4 de01 / 0±16 / 0 b00 / 0±01 / 0 4 d06 / 0±26 / 5 abc35 / 0±15 / 6 ef06 / 0±80 / 0 de21 / 0±70 / 3 e01 / 0±13 / 0 b00 / 0±01 / 0 5 b04 / 0±34 / 8 ab34 / 0±46 / 6 d03 / 0±93 / 0 d17 / 0±90 / 3 de01 / 0±17 / 0 b00 / 0±03 / 0 6 a14 / 0±09 / 9 ab36 / 0±80 / 6 bc03 / 0±23 / 1 g05 / 0±52 / 2 bc01 / 0±22 / 0 b00 / 0±02 / 0 7 m05 / 0±81 / 2 abc49 / 0±06 / 6 ef04 / 0±74 / 0 def12 / 0±38 / 3 de01 / 0±16 / 0 b00 / 0±01 / 0 8 k02.3±0.29 ab36 / 0±61 / 6 ef03 / 0±78 / 0 fg06 / 0±95 / 2 de01 / 0±16 / 0 b00 / 0±01 / 0 9 h03 / 0±49 / 3 abc60 / 0±27 / 6 de03 / 0±84 / 0 cd16 / 0±94 / 3 c01 / 0±18 / 0 b00 / 0±03 / 0 10 e05 / 0±58 / 4 a23 / 0±23 / 7 de05 / 0±85 / 0 fg06 / 0±93 / 2 bc01 / 0±22 / 0 b00 / 0±03 / 11 c08 / 0±94 / 6 ab56 / 0±89 / 6 c00 / 0±14 / 1 efg24 / 0±07 / 3 cd01 / 0±19 / 0 b 0.01±0.00 12 a04 / 0±91 / 8 abc07 / 0±30 / 6 b03 / 0±31 / 1 cd16 / 0±01 / 4 b01 / 0±26 / 0 b00 / 0±02 / 13 (witness) k02 / 0±11 / 3 c08 / 0±17 / 5 a04 / 0±43 / 1a 9.28±0.23 a 0.61±0.03 a 0.23±0.000 In each column, similar English letters indicate no significant difference (0.05). <P). Table 3- Digestibility of carbohydrates and proteins of different types of oak flour under laboratory conditions (standard error ± mean of three replicates) Treatment Carbohydrate digestibility (micrograms of maltose per milligram of sample) Protein digestibility (micrograms of tyrosine per milligram of sample) 1 b99 / 1±07 / 150 gh00 / 0±41 / 0 2 a09 / 0±76 / 166 e04 / 0±70 / 0 3 d46 / 0±76 / 107 f02 / 0±54 / 0 4 d87 / 0±42 / 105 e02 / 0±72 / 0 5 c97 / 0±86 / 134 a03 / 0±49 / 1 6 e11 / 1±79 / 99 e03 / 0±68 / 0 7 f56 / 0±08 / 70 g04 / 0±44 / 0 8 k80 / 0±56 / 42 h02 / 0±33 / 0 9 g46 / 0±16 / 67 f03.0±0.53 10 f24.1±71.71 d03.0±81.0 11 h58±94.065.0 c02.1±08.12 h72.0±54.58 b01.0±30.1 13 (witness) d72 / 0±24.108 f01 / 0±53 / 0 In each column, similar English letters indicate no significant difference (0.05). <P). A clear and precise statement of the advantages of the claimed invention over prior inventions. No invention has been registered in the field of fish food production using fermented acorns so far. In the only similar study conducted on carp by Naroui and Alamdari in 1401, published in the Iranian Journal of Fisheries, the use of fermented acorns did not have a significant negative effect on growth performance, survival rate, feed conversion ratio, feed intake rate, and protein and fat yield rates, but it caused a significant decrease in protein and fat reserves, as well as a decrease in carcass quality compared to the control treatment. In the study by Naroui and Alamdari in 1401, dry active baker's yeast (instant) was used at a dose of 1.5 grams per 100 grams of acorns in the diet of carp fish. However, in this invention, six different doses of yeast were studied in the form of 12 treatments and based on biochemical analysis and digestibility of acorns, and the decisive superiority of the dose of dry active baker's yeast (instant) equal to 10 grams per 100 grams of acorns was proven. Currently, fermented acorn flour is not used in the industry for making aquatic feed. Although yeast may be used in aquatic diets, this substance is inactivated in the process of making aquatic feed due to the use of high heat. Fermented acorn flour (using 10 grams of baker's yeast per 100 grams of acorn fruit) in the present invention for the production of fish food is the first to be introduced domestically and abroad and is a unique flour because its carbohydrate digestibility (1.24 times that of unfermented acorn) and its nutritional value in terms of protein (2.68 times that of unfermented acorn) and fat (1.25 times that of unfermented acorn) have significantly increased, but its total anti-nutritional phenolic compounds (2.38 times that of unfermented acorn) have decreased and it has the highest protein digestibility (approximately three times that of unfermented acorn) among fermented acorns. Given the large reserves of oak forests in Iran (more than 5 million hectares), which provide easy access to this primary food, the use of fermented oak in aquatic food increases the food security of the community and is also compatible with the digestive capacity of fish. Description of at least one implementation method for implementing the invention The acorn is collected from the forest. After manually removing the fruit cup and removing its outer shell with a knife or with the help of a mechanized device, the acorn kernel obtained is exposed to sunlight or placed in a fruit dryer. After drying, the inner shell of the fruit (the acorn) is removed manually. The acorn kernel is soaked in ordinary water for 24 hours. Soaking the acorns is done with a weight-volume ratio of 1 to 5 in an incubator at a temperature of 50 degrees Celsius and changing the water every 8 hours. After soaking, the acorns are ground using a meat grinder. Then, 10 grams of active dry baker's yeast (instant yeast) is added to them per 100 grams of acorns and dissolved in 50 milliliters of lukewarm ordinary water. Fermentation is done for 24 hours in an incubator at a temperature of 30 degrees Celsius. The fermented acorns are placed at 70°C for 10 minutes to inactivate the yeast. After inactivating the yeast, the fermented acorns are dried at 60°C. The acorns are then ground and sieved through a 250 micron sieve.This fermented oak flour can be used in aquatic feed factories as one of the raw materials in the production of carp fish food. Explicit mention of the industrial application of the invention It is possible to use fermented acorns under the specific conditions described in this invention and with a dose of instant baker's yeast (Saccharomyces cerevisiae) of 10 grams per 100 grams of acorns as one of the raw materials for the production of carp fish food in aquatic feed factories. Brief description of the invention The search for new food raw materials through various processing methods is of great importance in food security and the economy of society. More than 5 million hectares of Iran are covered with oak forests. In addition to having nutritious compounds, oak fruit contains a bitter and anti-nutritional substance called tannin. Anti-nutritional factors can be greatly reduced by various methods, including a combination of soaking (physical) and fermentation (biological).In this invention, through a new process, which includes: soaking acorn kernels without outer and inner shells in ordinary water with a weight-volume ratio of 1 to 5, placing the soaked acorn kernels in an incubator at 50°C for 24 hours and changing the water every 8 hours, washing the acorn kernels with ordinary water, grinding the acorn kernels, dissolving 10 grams of instant dry baker's yeast per 100 grams of acorns in 50 ml of lukewarm water, adding yeast solution to the ground acorn kernels, fermenting the acorns in an incubator for 24 hours at 30°C, inactivating the fermented acorns for 10 minutes at 70°C, drying the acorns by placing them at 60°C and then grinding them, acorn flour with high digestibility can be produced for use in carp diets, which is one of the It is the most important species of farmed fish in Iran and the world. In intensive aquaculture, sometimes up to 80 percent of the current costs are related to feed, and using the results of this invention will lead to increased food security and improved economic conditions for the community.

Claims

Complaint What is claimed: Claim 1) Processing of acorn flour by fermentation using baker's yeast, and in order: soaking acorn kernels without outer and inner shells in ordinary water with a weight-volume ratio of 1 to 5, placing the soaked acorn kernels in an incubator at 50°C for 24 hours and changing the water every 8 hours, washing the acorn kernels with ordinary water, grinding the acorn kernels, dissolving instant dry baker's yeast at a rate of 10 grams per 100 grams of acorns in 50 ml of lukewarm water, adding yeast solution to the ground acorn kernels, fermenting the acorns in an incubator for 24 hours at 30°C, placing the fermented acorns at 70°C for 10 minutes to inactivate the yeast, drying the acorns at 60°C, and finally grinding the acorns to obtain flour with Particle sizes smaller than 250 microns enhance its nutritional value.

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