CN105231000B - Method for producing antibacterial nutritional compound feed additive by using aquatic product leftovers - Google Patents
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Abstract
The invention belongs to the technical field of feed additives, and discloses a method for producing an antibacterial nutritional compound feed additive by using aquatic product leftovers, which takes aquatic product processing leftovers as main raw materials, is supplemented with feeding plant protein, bran and bagasse (or rice hulls), is inoculated with a bacillus natto NT-6 strain for solid state fermentation, and is combined with a secondary enzymolysis regulation and control technology, so that the prepared feed additive can be used as a substitute of feeding antibiotics and other growth promoters, is used for improving the production performance, the immunity, the disease resistance and the feed conversion rate of animals, and can also be used for preventing and treating various diseases in the animal breeding process. The method can simplify the production process of the active peptide for feed, remarkably reduce the product cost, improve the product efficacy, expand the product application range, improve the added value of leftovers, realize the slag-free discharge efficient environment-friendly industrial production of the active peptide for feed, and promote the healthy sustainable development of the aquaculture industry.
Description
Technical Field
The invention relates to the technical field of feed additives, in particular to a method for producing an antibacterial nutritional compound feed additive by using aquatic product leftovers.
Background
The method has the advantages of huge amount of aquatic product processing leftovers, low recycling level, serious waste and environmental pollution, and urgent need to search a new reprocessing method with high added value and low emission. The active peptide for feed is a high-value and low-threshold product, and becomes a new way for developing and utilizing the marine low-value protein. Antibacterial peptides and nutritional small peptides are considered to be the most potential feed antibiotic substitutes at present as major members of active feed peptides.
The bacillus antibacterial lipopeptide is one of the antibacterial peptide families, and the members of the bacillus antibacterial lipopeptide are usually surfactant (surfactin), fungicin (fengycin), iturin (iturin), bacillomycin (bacillus), antimycobacterial subtilisin (mycosubtilin), plipastatin (plipstatin) and the like. The antibacterial peptide has remarkable antibacterial, mildew, virus and protozoon resisting effects, has very good stability to acid-base and heat treatment, and is safe to take orally; the characteristics contribute to the wide application of the feed additive industry. Part of members of the bacillus antibacterial lipopeptide are applied to disease resistance and growth promotion of animal breeding, and the effect is obvious. But the lipopeptide producing capability of the strain is relatively low in the application process; the antibacterial spectrum of the product is relatively narrow, and the antibacterial pertinence is single; low fermentation yield and the like. Especially, the liquid fermentation production of the bacillus antibacterial lipopeptide can generate a large amount of foam, so the tank efficiency is low, and the method becomes the bottleneck of industrialization.
The small feed peptide is an intermediate product of proteolysis and is a compound consisting of 2 or more than 2 amino acid residues. The nutritional small peptide has high absorptivity, high conversion rate and immunostimulation effect, and can significantly improve the survival rate of animals, promote the absorption and utilization of mineral elements in feed and the conversion of feed, promote the synthesis of protein in the bodies of the animals and enhance the autoimmunity of the animals when added into the word material, thereby realizing the substantial yield increase and the synergism of the cultivation. At present, the main raw materials of the active nutritional small peptide for feed use adopt protein resources such as soybean meal, feather meal, fish meal and the like, and are produced by an enzymolysis method, a microbial fermentation method or a combination of the enzymolysis method and the microbial fermentation method. Many enterprises are studied at home and abroad, the products are applied in a certain range, but the defects of low yield, high cost, single function and the like exist, and further large-range popularization is limited.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for producing an antibacterial nutritional compound feed additive by using aquatic product leftovers.
The second purpose of the invention is to provide the antibacterial nutritional compound feed additive obtained by the method.
A third object of the invention is to provide the use of said additive.
The purpose of the invention is realized by the following technical scheme:
a method for producing an antibacterial nutritional compound feed additive by using aquatic product processing leftovers comprises the following steps:
s1, preparing a fermentation ingredient: mincing aquatic product leftovers, adding plant feed protein, bran, bagasse or rice hulls, and mixing the materials in a mass ratio of 4-8: 1: 1: 0.1-0.4, mixing to obtain a base material, and then adding auxiliary materials and water; obtaining a fermentation ingredient after stewing and cooling;
s2, solid fermentation: when the temperature of the fermentation ingredient of S1 is 40 ℃, inoculating the seed liquid of Bacillus natto NT-6, fermenting at 25-35 ℃, controlling the air humidity at 75-85%, and stopping fermentation after 36-72 h;
s3, secondary enzymolysis: transferring the fermented material into a heat preservation device, raising the temperature to 45-55 ℃ at a temperature rise rate of 3-5 ℃/30min, preserving the heat for 2-5 h, finally drying, controlling the moisture of the dried material within 12%, and crushing to obtain the fertilizer.
In the prior art, fermentation of antibacterial lipopeptide is separated from fermentation of nutritional small peptide, and the existing process for producing the feed active peptide by using aquatic product leftovers has low yield, high cost and relatively single product function; the production cost of the antibacterial lipopeptide is high, the antibacterial spectrum is narrow, and because the production processes of the two substances in the prior art have some defects, if the fermentation production of the antibacterial lipopeptide and the enzymolysis process of the nutritional small peptide are combined, the antibacterial lipopeptide can be produced by using aquatic product leftover low-value protein as a nutritional substrate of bacillus, and the nutritional small peptide can be produced by using compound protease generated by the bacillus in the fermentation process to perform enzymolysis on the leftover low-value protein; on the basis of the above thought, the applicant provides a production method for producing the composite antibacterial lipopeptide and the small nutrient peptide, namely, the solid state fermentation is optimized, secondary enzymolysis is carried out on the basis of the solid state fermentation, and the product obtained after the secondary enzymolysis is rich in the antibacterial lipopeptide, the small nutrient peptide, the complex enzyme and the bacillus probiotics.
Preferably, the cooking temperature reduction of S1 is: introducing steam under the pressure of 0.2Mpa for cooking for 15-20 min, and cooling the material to 25-40 ℃ after material steaming.
It is emphasized that the solid fermentation and secondary enzymolysis of the present invention are combined together to ensure that the final product is rich in antibacterial lipopeptide, small nutrient peptide, complex enzyme and bacillus probiotics.
Preferably, the inoculation amount of the bacillus natto NT-6 seed liquid is 0.1-5% of the mass of the fermentation ingredients.
Preferably, the aquatic product leftovers of S1 are tilapia, cod, scallop skirt, silver carp, green scale fish, grass carp, bonito or shrimp.
The fermentation strain used in the invention is Bacillus natto NT-6, also called Bacillus subtilis subsp. natto (Bacillus subtilis subsp. natto) NT-6 strain, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.8121 and the preservation address: west road No. 1, north west of the facing yang district, beijing, institute of microbiology, academy of sciences of china, with a date of collection of 2013, 9 months and 3 days.
The preparation process of the bacillus natto NT-6 seed liquid is as follows:
(1) seed medium (g/L): 10.0 parts of tryptone, 5.0 parts of yeast extract, 5.0 parts of NaCl, 1000mL of water and 7.2 parts of pHs;
(2) and (3) expanding and culturing seeds: gradually carrying out amplification culture on the Bacillus natto NT-6 strain, inoculating the Bacillus natto NT-6 strain to a seed culture medium, and carrying out aeration culture for 18h for later use.
The antibacterial lipopeptide production strain natto bacteria NT-6 used by the invention has obvious advantages in antibacterial activity and antibacterial spectrum compared with antibacterial lipopeptides produced by other bacilli. The lipopeptide component produced by the strain is much richer than that of related strains for producing the antibacterial lipopeptide reported at present, and the produced antibacterial lipopeptide component contains more than twenty lipopeptide analogues, which is extremely rare in the report of the existing lipopeptide producing strains. The perfect synergistic effect of the three groups of lipopeptide homologues ensures that the composite lipopeptide produced by the strain has obvious inhibition effect on most tested gram-negative and gram-positive bacteria and moulds. In addition, the antibacterial lipopeptide has good surface activity, so that the microbes are difficult to generate drug resistance and have good antiviral and antiprotozoal effects. Can be used for preventing and treating various diseases (such as drug-resistant bacterial diarrhea, coccidiosis, porcine parvovirus disease, avian influenza and the like) in the animal breeding process, and greatly expands the application range of the feed additive prepared by the invention.
Preferably, the plant feed protein of S1 is one or two of soybean meal, detoxified rapeseed meal, detoxified cottonseed meal, and peanut meal.
Preferably, the auxiliary materials are molasses or glucose, sodium glutamate, dipotassium hydrogen phosphate and MgSO4·7H2And O. Specifically, the auxiliary materials comprise 1-5% of molasses (or glucose), 0.1-0.5% of sodium glutamate, 0.05-0.5% of dipotassium hydrogen phosphate and 0.1-0.3% of MgSO (MgSO) based on the weight of the base material4·7H2O。
The invention provides an antibacterial nutritional compound feed additive obtained by the method; the feed additive contains composite antibacterial lipopeptide, crude protein, polypeptide, bacillus natto, xylanase, protease and amylase.
Specifically, the content of the composite antibacterial lipopeptide is 2-20mg/g, 10-30% of polypeptide and 1.0-5.0 x 10% of bacillus natto9cfu/g, xylanase activity of 5000-8000U/g, protease activity of 600-1000U/g and amylase activity of 4000-8000U/g.
More preferably, the content of crude protein in the compound feed additive obtained from the leftovers used in the invention is 30-60%.
The feed active peptide is produced by solid state fermentation of high-yield and excellent antibacterial lipopeptide natto bacillus, the product is rich in high-activity antibacterial lipopeptide and a large amount of nutritional small peptides, and meanwhile, the final product also contains rich compound enzyme systems and beneficial-growth bacillus natto. The feed disease prevention growth promoting agents such as the composite antibacterial lipopeptide, the nutritional small peptide, the composite enzyme, the bacillus probiotics and the like are produced in a combined mode, so that a high-concentration antibacterial lipopeptide product can be obtained, the problem of a biological enzyme source required by protein enzymolysis in the production of the nutritional small peptide can be solved, and the produced product has the functions of the antibacterial lipopeptide and the effect of the nutritional small peptide; meanwhile, due to the existence of the antibacterial lipopeptide in the matrix, the putrefaction problem of the enzymolysis process of leftover raw materials can be avoided, and the enzymolysis process can be innovated by adopting a raw material sterilization-free production mode.
The composite antibacterial lipopeptide consists of part or all of components in three antibacterial lipopeptide homologs of iturins, fengycins and surfactin. Wherein the iturins contains homologue components having molecular weights of 1043.2, 1057.2, 1071.0, 1071.0 Da; fengycins contain homolog components with molecular weights of 1491.7, 1492.5, 1505.7, 1519.7 Da; surfactin contains homolog components with molecular weights of 994.3, 1009.0, 1008.8, 1009.9, 1008.7, 1022.7, 1022.6, 1036.8, 1036.5, 1036.6, 1051.8, 1050.6, 1051.2, 1051.6, 1065.8, 1080.2 Da.
The invention also provides application of the antibacterial nutritional compound feed additive in animal disease resistance.
The invention also provides application of the antibacterial nutritional compound feed additive in promoting animal growth.
The feed additive can be directly added into feed or prepared into granular feed, and is used for disease resistance and growth promotion of pigs, cattle, sheep, poultry and aquatic animals.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for producing an antibacterial and nutritional compound feed additive by using aquatic product processing leftovers, which is characterized in that the aquatic product processing leftovers are used as main raw materials, feeding plant protein, bran and bagasse (or rice hulls) are used as auxiliary materials, bacillus natto NT-6 strain is inoculated, solid state fermentation is carried out, and a secondary enzymolysis regulation and control technology is combined to produce the compound feed additive rich in high-activity compound antibacterial lipopeptide, nutritional small peptide, compound enzyme and bacillus probiotics; in addition, the fermentation equipment is simple, the concentration of the unit matrix is high, the yield is high, and the problem of foam which is difficult to solve in the process of producing the antibacterial lipopeptide by large-scale liquid fermentation can be avoided in production; the method simplifies the process, greatly reduces the cost, expands the application range of the product and enhances the competitiveness of the product; the added value of the leftovers can be obviously improved, the high-efficiency environment-friendly industrial production without slag discharge is realized, and the sustainable development of the aquaculture industry is promoted.
Detailed Description
The following examples are presented to further illustrate the present invention and should not be construed as limiting the invention. It is within the scope of the present invention to make simple modifications or alterations to the methods, procedures or conditions of the present invention without departing from the spirit and substance of the invention; unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
The method for preparing the antibacterial nutritional compound feed additive from the leftovers of the fresh tilapia comprises the following steps:
s1, preparing a fermentation ingredient: mincing fresh tilapia mossambica (viscera removed) leftovers, adding bean pulp, bran and bagasse (sieved by a sieve with less than 60 meshes) according to a mass ratio of 4: 1: 1: 0.2 mixing to obtainAdding 1% of molasses, 0.5% of sodium glutamate, 0.05% of dipotassium hydrogen phosphate and 0.1% of MgSO (MgSO) into the base material4·7H2O and 60% water, placing the prepared materials into a rotary steamer, and steaming for 15min under the pressure of 0.2 Mpa. When the temperature of the material is reduced to 40 ℃ after the material steaming, the final water content is kept at 70 percent.
S2, solid fermentation: when the temperature of the materials is 30 ℃, inoculating the bacillus natto seed liquid which is subjected to enlarged culture according to the mass percent of 1 percent, and uniformly mixing. And (3) conveying the inoculated materials to a fermentation workshop ventilation fermentation tank (bed) with the temperature controlled at 25 ℃ for fermentation culture. The air humidity of the fermentation chamber is controlled at 80%, and fermentation is stopped after fermentation culture for 36 h.
S3, secondary enzymolysis: transferring the fermented material to a heat preservation device, raising the temperature to 50 ℃ at the temperature rise rate of 5 ℃/30min, and then keeping for 2 h. After the heat preservation process is finished, the fermented material is conveyed to crawler-type drying equipment to be dried at low temperature, and the moisture of the dried material is controlled within 11%. After being crushed, the composite feed additive product rich in antibacterial lipopeptide, nutrient peptide, complex enzyme and bacillus probiotics is obtained.
The main indexes of the effective components of the composite feed additive produced in the steps are as follows: the content of the composite antibacterial lipopeptide is 5-10 mg/g (calculated by LC-MS detection method), the content of crude protein is more than 50%, the content of polypeptide is more than 20%, and the content of bacillus natto is 1.0-5.0 multiplied by 109cfu/g, xylanase activity is more than 6000U/g, protease activity is more than 800U/g, and amylase activity is more than 6000U/g.
Example 2
The method for preparing the antibacterial nutritional compound feed additive from the leftovers of the fresh prawn heads comprises the following steps:
s1, preparing a fermentation ingredient: mincing fresh prawn heads, adding bean pulp, bran and bagasse (less than 60 meshes) according to a mass ratio of 8: 1: 1: 0.3, adding 4 percent of molasses, 0.1 percent of sodium glutamate, 0.05 percent of dipotassium hydrogen phosphate and 0.1 percent of MgSO (MgSO) based on the weight of the base material4·7H2O and 65% water, placing the prepared materials into a rotary steamer, and steaming for 18min under the pressure of 0.2 Mpa. After steaming the materialWhen the temperature of the material is reduced to 40 ℃, the final water content is kept at about 70 percent.
S2, solid fermentation: when the temperature of the prepared fermentation material is 35 ℃, inoculating the bacillus natto seed liquid which is subjected to enlarged culture according to the mass percentage of 2 percent, and uniformly mixing. And (3) conveying the inoculated materials to a fermentation workshop ventilation fermentation tank (bed) with the temperature controlled at 30 ℃ for fermentation culture. The air humidity of the fermentation chamber is controlled at 85%, and fermentation is stopped after fermentation culture for 48 h.
S3, secondary enzymolysis: transferring the fermented material to a heat preservation device, raising the temperature to 55 ℃ at the temperature rise rate of 5 ℃/30min, and then keeping the temperature for 3.5 h. After the heat preservation process is finished, the fermented material is conveyed to crawler-type drying equipment to be dried at low temperature, and the moisture of the dried material is controlled within 11%. After being crushed, the composite feed additive product rich in composite antibacterial lipopeptide, nutrient peptide, composite enzyme and bacillus probiotics is obtained.
The main indexes of the effective components of the composite feed additive produced in the steps are as follows: the content of the composite antibacterial lipopeptide is more than 10mg/g (calculated by LC-MS detection method), the content of crude protein is more than 60%, the content of polypeptide is more than 25%, and the content of bacillus natto is 1.0-5.0 multiplied by 109cfu/g, xylanase activity is more than 3000U/g, protease activity is more than 1000U/g, and amylase activity is more than 4000U/g.
Example 3
The method for preparing the antibacterial nutritional compound feed additive from the leftovers of the skirt of the fresh scallop comprises the following steps:
s1, preparing a fermentation ingredient: mincing fresh scallop skirt, adding rapeseed dregs, bran and rice hulls according to a mass ratio of 5: 1: 1: 0.2, then adding 2 percent of molasses, 0.2 percent of sodium glutamate, 0.06 percent of dipotassium hydrogen phosphate and 0.2 percent of MgSO 2 percent of the weight of the base material4·7H2O and 65% water, placing the prepared materials into a rotary steamer, and steaming for 20min under the pressure of 0.2 Mpa. When the temperature of the material is reduced to 40 ℃ after the material steaming, the final water content is kept at 72 percent.
S2, solid fermentation: when the temperature of the prepared fermentation material is 35 ℃, inoculating the bacillus natto seed liquid which is subjected to enlarged culture according to the mass percentage of 2 percent, and uniformly mixing. And (3) conveying the inoculated materials to a fermentation workshop ventilation fermentation tank (bed) with the temperature controlled at 32 ℃ for fermentation culture. The air humidity of the fermentation chamber is controlled at 85%, and the fermentation is stopped after fermentation culture for 72 h.
S3, secondary enzymolysis: transferring the fermented material to a heat preservation device, raising the temperature to 45 ℃ at the temperature rise rate of 5 ℃/30min, and then keeping for 5 h. After the heat preservation process is finished, the fermented material is conveyed to crawler-type drying equipment to be dried at low temperature, and the moisture of the dried material is controlled within 12%. After being crushed, the composite feed additive product rich in composite antibacterial lipopeptide, nutrient peptide, composite enzyme and bacillus probiotics is obtained.
The main indexes of the effective components of the composite feed additive produced by the method are as follows: the content of the composite antibacterial lipopeptide is more than 5mg/g (calculated by LC-MS detection method), the content of crude protein is more than 40%, the content of polypeptide is more than 15%, and the content of bacillus natto is 1.0-5.0 multiplied by 109cfu/g, xylanase activity is more than 4000U/g, protease activity is more than 700U/g, and amylase activity is more than 4000U/g.
Comparative example 1
The experimental method is the same as that of example 2, and the only difference is that the secondary enzymolysis in S3 is carried out according to the following operations: transferring the fermented material to a heat preservation device, raising the temperature to 60 ℃ at the temperature rise rate of 2 ℃/30min, and then keeping for 5 h. After the heat preservation process is finished, the fermented material is conveyed to crawler-type drying equipment to be dried at low temperature, and the moisture of the dried material is controlled within 11%. The obtained product is detected by crushing, and the main indexes of the effective components are as follows: the content of composite antibacterial lipopeptide is 1mg/g (calculated by LC-MS detection method), the content of crude protein is 40%, the content of polypeptide is 10%, the content of Bacillus natto is 2.4 multiplied by 107cfu/g, xylanase activity of 86U/g, protease activity of 58U/g and amylase activity of 200U/g.
Comparative example 2
The experimental method is the same as that in the example 2, the only difference is that in the S1, the fresh scallop skirt is minced, and the rapeseed dregs, the bran and the rice hull are mixed according to the mass ratio of 3: 1: 1: 0.5, and finally detecting the obtained product, wherein the main indexes of the effective components are as follows: composite antibacterial lipopeptide containing5mg/g (calculated by LC-MS detection method), crude protein content of 20%, polypeptide content of 4%, and Bacillus natto content of 3.5 × 109cfu/g, xylanase activity of 5000U/g, protease activity of 800U/g and amylase activity of 6000U/g.
Comparative example 3
The experimental method is the same as that of example 2, the only difference is that S2 solid fermentation is performed at 20 ℃, the air humidity is controlled at 70%, the fermentation is stopped after 24h, then the enzymolysis is performed, finally the obtained product is detected, and the main indexes of the effective components are as follows: the content of composite antibacterial lipopeptide is 0.3mg/g (calculated by LC-MS detection method), the content of crude protein is 40%, the content of polypeptide is 1.5%, the content of Bacillus natto is 2.0 × 106cfu/g, xylanase activity of 100U/g, protease activity of 65U/g and amylase activity of 100U/g.
Claims (9)
1. A method for producing an antibacterial nutritional compound feed additive by using aquatic product processing leftovers is characterized by comprising the following steps:
s1, preparing a fermentation ingredient: mincing aquatic product leftovers, adding plant feed protein, bran, bagasse or rice hulls, and mixing the materials in a mass ratio of 4-8: 1: 1: 0.1-0.4, mixing to obtain a base material, and then adding auxiliary materials and water; obtaining a fermentation ingredient after stewing and cooling;
s2, solid fermentation: when the temperature of the fermentation ingredient of S1 is 40 ℃, inoculating a Bacillus natto NT-6 seed solution, wherein the inoculation amount of the Bacillus natto NT-6 seed solution is 0.1-5% of the mass of the fermentation ingredient, fermenting at 25-35 ℃, controlling the air humidity at 75-85%, and stopping fermentation after 36-72 h;
s3, secondary enzymolysis: transferring the fermented material into a heat preservation device, raising the temperature to 45-55 ℃ at a temperature rise rate of 3-5 ℃/30min, preserving the heat for 2-5 h, finally drying, controlling the moisture of the dried material within 12%, and crushing to obtain the fertilizer.
2. The method for producing an antibacterial nutritional compound feed additive from aquatic product processing leftovers according to claim 1, wherein the aquatic product leftovers in S1 are leftovers of tilapia, cod, scallop, silver carp, green fish, grass carp, bonito, or shrimp.
3. The method for producing the antibacterial nutritional compound feed additive by using the aquatic product processing leftovers according to claim 1, wherein the plant feed protein of S1 is one or two of soybean meal, detoxified rapeseed meal, detoxified cottonseed meal and peanut meal.
4. The method for producing the antibacterial nutritional compound feed additive by using the aquatic product processing leftovers according to claim 1, wherein the auxiliary materials are molasses or glucose, sodium glutamate, dipotassium hydrogen phosphate and MgSO4·7H2O。
5. An antibacterial nutritional compound feed additive obtained by the method of any one of claims 1 to 4.
6. The antibacterial nutritional compound feed additive according to claim 5, wherein the feed additive comprises a compound antibacterial lipopeptide, crude protein, polypeptide, Bacillus natto, xylanase, protease and amylase.
7. The antibacterial nutritional compound feed additive according to claim 6, wherein the content of the compound antibacterial lipopeptide is 2-20 mg/g, the content of the polypeptide is 10-30%, the content of bacillus natto is 1.0-5.0 x 109cfu/g, the xylanase activity is 5000-8000U/g, the protease activity is 600-1000U/g, and the amylase activity is 4000-8000U/g.
8. The use of the antibacterial nutritional compound feed additive of claim 5 in the preparation of feed with animal disease-resistant effect.
9. Use of the antibacterial nutritional compound feed additive according to claim 5 for promoting the growth of animals.
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| CN111587949A (en) * | 2020-05-26 | 2020-08-28 | 广东海洋大学 | Application of bacillus natto NT-6 in degradation of antibiotics in meat and bone meal of sick animals |
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| CN102321182A (en) * | 2011-09-22 | 2012-01-18 | 广东海洋大学 | Composite antibiosis peptide and working method thereof that bacillus natto NT-6 bacterial strain is produced |
| CN102550804A (en) * | 2012-02-04 | 2012-07-11 | 广东海洋大学 | Method for preparing feed protein by mixed fermentation of tilapia leftovers and soybean meals |
| CN103283961A (en) * | 2013-05-10 | 2013-09-11 | 浙江工业大学 | Method for preparing health-benefiting fermented soybean meal |
| CN104686804A (en) * | 2015-03-02 | 2015-06-10 | 广东海洋大学 | Method for preparing active peptide feed additive from aquatic product leftovers |
| CN104719639A (en) * | 2015-03-02 | 2015-06-24 | 广东海洋大学 | Method for preparing antibacterial feed protein by utilizing aquatic product leftovers |
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