CN110419557A - A method of accelerating beef cattle postmortem aging - Google Patents

Abstract

The invention discloses a kind of methods for accelerating beef cattle postmortem aging.Described method includes following steps: keeping beef mature by the way of heel string hanging-Jingbi constraint, and then improves beef quality；The mode of the heel string hanging-Jingbi constraint refers to hanging beef side body using by the way of heel string hanging, then applies radial pull between distal forearm and ox neck；The size of the radial pull is 10~50KG；The beef quality refers to the tenderness of beef.The present invention is able to suppress longissimus dorsi muscle and shrinks during cadaveric rigidity, makes back leg and along spinal muscular in stretching and have the state slightly stretched, it is possible to reduce longissimus dorsi muscle contraction of muscle, increase length of sarcomere, reduce diameter of muscle fiber, product tenderness is good, improves the consistency of meat tenderness degree.

Description

A method of accelerating beef cattle postmortem aging

Technical field

The present invention relates to a kind of methods for accelerating beef cattle postmortem aging, and in particular to a kind of to use heel string hanging-Jingbi beam The method that the mode tied up accelerates beef cattle postmortem aging.

Background technique

The edible quality of meat determines the value of commodity, in many quality factors of meat, tenderness be consumer the most The edible quality of concern.Tenderness of beef utilizing is low, tenderness consistency difference is the two big main problems that current meat industry faces between product, So it is very necessary to improve tenderness of beef utilizing.The factor for influencing tenderness mainly includes killing preceding factor (kind and genotype, Animal Sex With the age, raising and kill before management etc.) with kill after factor (butcher trunk processing in muscle pH value and temperature fall off rate, electricity Stimulation process, trunk suspending way, the maturation of meat and cooking method etc.), extending maturation time can solve Chinese sirloin tenderness The problem of difference.But maturation time extension will greatly increase enterprise's production cost, cause the waste of energy and resource.Therefore, such as What quickly improves tenderness of beef utilizing, shortens the problem of beef maturation time is China Meat industry urgent need to resolve.

Summary of the invention

The object of the present invention is to provide a kind of sides for accelerating beef cattle postmortem aging by the way of heel string hanging-Jingbi constraint Method (is usually chosen between ox atlas and the second neck) between ox distal forearm and ox neck on the basis of heel string is hung and is added One radial pull is allowed to tighten, which is able to suppress longissimus dorsi muscle and shrinks during cadaveric rigidity, makes back leg and along backbone Muscle is in stretch and have the state slightly stretched, it is possible to reduce longissimus dorsi muscle contraction of muscle increases length of sarcomere, reduces muscle fibre Diameter, product tenderness is good, improves the consistency of meat tenderness degree.

Specifically, the method provided by the present invention for accelerating beef cattle postmortem aging speed, includes the following steps:

Keep beef mature (acid discharge) by the way of heel string hanging-Jingbi constraint, and then accelerates beef cattle postmortem aging speed；

The mode of the heel string hanging-Jingbi constraint refers to hanging beef side body using by the way of heel string hanging, then Apply radial pull between distal forearm and ox neck (usually choosing between ox atlas and axis).

In above-mentioned method, the size of the radial pull can be 10~50KG.

In above-mentioned method, the beef cattle postmortem aging speed refers to the increased speed of the tenderness of beef.

In above-mentioned method, the hanging time is 1h to 21 days.

Heel string hanging is traditional suspending way of ox trunk, and this suspending way makes semimembranosus, semitendinosus, the stock two of ox The posterior musculars such as flesh and longissimus dorsi muscle, in dissociating relatively and being easy to shrink the state for becoming tough, reduce bone during cadaveric rigidity To back leg and along the restriction effect of spinal muscular.The suspending way is weaker to the stretching of backbone, and backbone is in bending state, reduces The inhibition that longissimus dorsi muscle position meat is shunk during stiff.The present invention is by using heel string hanging-Jingbi constraint mode Solves the above problem.

The present invention has investigated heel string hanging-influence of the Jingbi constraint processing mode to beef cattle postmortem aging speed, chooses 6 Male Xinjiang rivers similar in age and liveweight, half trunk of right side hangs mature mode using common heel string after butchering, left Half trunk of side is mature using heel string hanging Jingbi constraint technical treatment.After government official 1 hour and 72 hours sampling determination pH value, The indexs such as cooking loss rate, shearing force, muscle fibril snip to change exponent, and by transmission electron microscope, protein science research with regard to its machine Reason is studied.Investigate as a result, it has been found that, 3 days after being killed using the sample that heel string hanging-Jingbi constraint mode is handled compared with control sample Shear force value significantly reduce, muscle fibril snip to change exponent obviously rises；0h is identified by iTRAQ protein group quantitative technique Totally 2608, the protein of the brown ox back longissimus of different suspending ways with 72h's, between BZC1VSBZC2, BZC3VSBZC4 group The differentially expressed protein compared has 20,12 respectively.The protein that identifies carry out functional annotation be mostly cell Proliferation, Metabolic activity, biological cycle etc.；Carrying out the functional annotation overwhelming majority to the differentially expressed protein of comparison among groups is all energy Conversion, biosynthesis, signal transduction mechanism etc..The GO of all protein identified and comparison among groups differential expression protein, Pathway access enrichment, wherein there is many accesses related to meat tenderness degree according to the present invention.The above results show that heel string is hung Extension-Jingbi constraint mode can greatly improve beef cattle postmortem aging speed, shorten beef maturation time, have compared with senior engineer The value that industry is promoted.

Detailed description of the invention

Fig. 1 is the beef longissimus dorsi muscle shear force value (n=6) under different suspending ways and maturation time.

Fig. 2 is different suspending ways and maturation time beef longissimus dorsi muscle fiber Change of Ultrastructure.

Fig. 3 is protein concentration canonical plotting.

Fig. 4 is SDS-PAGE (polyacrylamide gel) electrophoresis detection result.

Fig. 5 is the significant difference protein profile that different suspending way and maturation time compare.

Fig. 6 is all albumen KOG annotated maps identified.

Fig. 7 is all protein G O annotations identified.

Fig. 8 is all protein Pathway annotations identified.

Fig. 9 is the enrichment point of BZC1 and BZC2 group differentially expressed protein in biological process, cellular component and molecular function Analysis.

Figure 10 is the enrichment point of BZC3 and BZC4 group differentially expressed protein in biological process, cellular component and molecular function Analysis.

Figure 11 is to lower statistical chart in BZC1-BZC2 differential protein Pathway classification.

Figure 12 is to lower statistical chart in BZC3-BZC4 differential protein Pathway classification.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1,

1, materials and methods

1.1 test material

Beef sample is derived from Xinjiang Yili Ning County Yi Xin cattle and sheep cultivation Specialty Co-operative Organization.

Choose 6 30 monthly age Xinjiang rivers bulls (liveweight is (566 ± 32) kg), after butchering segmentation, right half trunk Mature using common heel string suspending way, left half trunk is mature using Jingbi constraint suspending way, i.e., in ox distal forearm and ox First and second cervical vertebra junction applies the radial pull of 30KG, and 1h and 72h takes longissimus dorsi muscle sample after government official respectively, according to various The processing of specific targets testing requirements, storage sample.

Test reagent: potassium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, sodium azide, ethylene glycol-is bis--(2- ammonia Base ether) tetraacethyl (EGTA), sodium hydroxide, sodium potassium tartrate tetrahydrate, sodium hydroxide, copper sulphate, methanol, ethyl alcohol, acetone, high chlorine Acid, glutaraldehyde, vinegar lead, osmic acid, trypsase, bovine serum albumin(BSA) BSA standard items.The above reagent removes trypsase and cow's serum Albumin standard is that other are AR grades outside BR grades.

1.2 test apparatus

FJ200-S type high-shear homogenizer (Beijing Wei Xinyiao Science and Technology Ltd.)；LG10-24A type supercentrifuge (north Jing Jingli centrifuge Co., Ltd)；XS105 type electronic balance (Mei Tele-support benefit Instrument Ltd.)；Visible point of H721 type Light photometer (Tianjin Jim Press Instrument Ltd.)；TA-XT2i Texture instrument (Stable Micro System company, Britain)； DK-S28 electric-heated thermostatic water bath (the upper macro experimental facilities Co., Ltd of Nereid)；H-7500 type transmission electron microscope (Japanese day Vertical company)；Gel imaging system (Bio-Rad company, the U.S.)；High-pressure sterilizing pot (TOMY company)；Ice machine ( AF100)；Ultra low temperature freezer (Thermo company)；YDS-30-125 type liquid nitrogen container (Sichuan East Asia liquid nitrogen tank farms)；Ultrapure water instrument (Milli-Synthesis, MILLIPORE)；Multi-function microplate reader (Thermo company)；Adjustable micropipettor (Eppendorf company)；Thermostat water bath (DHW-6002, Beijing Ai Qixia commerce trading center)；SCIENTZ-48 type high throughput group Knit dismembyator (NingBo XinZhi Biology Science Co., Ltd).

1.3 test method

1.3.1pH measurement

It is measured with reference to GB9695.5-2008 meat and meat products pH value, weighs the rubbing of 4g meat sample, be dissolved in the KCl of 40ml0.1M Solution (into 1000ml), with the homogenizer homogeneous of 20000r/min, survey 7.5g potassium chloride constant volume while stirring by magnetic stirring apparatus PH value.

1.3.2 shearing force measures

Referring to Zhang Yuqing [Zhang Yuqing .2015] method.Longissimus dorsi muscle meat sample is taken, 5cm × 5cm × 3cm meat piece is cut, With shortening until central temperature takes out meat piece after reaching 70 DEG C naturally cools to room in 80 DEG C of water-baths after retort pouch environmental sealing Warm (20 DEG C), each temperature each time point take 6 samples, and each sample is flat with diameter 1.27cm sampler along muscle fibre direction Row takes 3 cedductors, with its shearing force of instrumental test, as a result takes its average value.

1.3.3 cooking loss rate measures

After taking out measurement meat sample, after claiming meat piece quality W1, after meat piece sealing packaging, it is heated in meat piece in 80 DEG C of water-baths Heart temperature reaches 70 DEG C, is then put in 0~4 DEG C overnight, blots meat piece surface juice with filter paper, claims meat piece quality, be denoted as W2；It presses Formula calculates cooking loss rate (%).

Cooking loss rate=(W1-W2)/W1 × 100%

1.3.4 muscle fibril snip to change exponent

Referring to the measuring method of yellow bright [Huang Ming, 2003]: 0d and 3d processing and untreated meat sample after taking longissimus dorsi muscle to kill It is each appropriate, accurately weigh 2g after removing visible fat, be added 20mL be pre-chilled to 2 DEG C MFI buffer (KCl containing 100mmol/L, 11.2mmol/L K₂HPO₄、8.8mmol/L KH₂PO₄, 1mmol/L ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) (EGTA), 1mmol/L MgCl₂With 1mmol/L NaN₃), it is homogenized 3 times, each 20s, midfeather 1min in homogenizer high speed.Homogenate is in 4 DEG C of items 10000r/min is centrifuged 15min under part, discards supernatant liquid.20mL buffer is added in precipitating to be homogenized again, then is centrifuged and discards Clear liquid.5mL buffer is added in precipitating to be homogenized, filters the suspension with 200 mesh nylon screens, removes connective tissue, then Centrifuge tube is washed with the buffer of 5 times of volumes and is filtered, merging filtrate, as fribrillin solution.Resulting muscle fibril Albumen suspension measures its protein content with biuret method, is then adjusted its mass concentration to 0.5mg/mL with MFI buffer, Its absorbance is measured at 540nm, institute's value is multiplied by 200, as muscle fibril snip to change exponent (MFI).

1.3.5 transmission electron microscope observing microstructure

With reference to Li et al. (Li, et al., 2013) method and be modified slightly.0d and 3d after government official is taken to handle and untreated sample Product are cut into 3 × 1 × 1mm with knife blade³Small item, it is more than hour to be fixed 4 for musculature with glutaraldehyde fixer.So It is handled afterwards by following procedure:

(1) flushing, fixation, dehydration of sample, replace and be impregnated with: after glutaraldehyde is fixed, with 0.1M phosphate buffer solution (pH 7.4) 2h is rinsed.Evacuation cleaning solution quickly and covers 1% osmic acid down to centrifuge tube to sample is not crossed in ventilation at once Lid.Gently vibrating example keeps sample fixed sufficiently, fixed 2h.Then each with a series of ethanol solution serial dehydration of concentration Spacing gradient 6min.It after dehydrated alcohol dehydration, is replaced 6 times, each 3min with anhydrous propanone, room temperature carries out.Then acetone is used: tree The mixing that rouge=2:1 ratio is prepared is impregnated with liquid, in 35 DEG C of insulating boxs, is impregnated with 2h.It is impregnated with overnight with embedding liquid in 35 DEG C of insulating boxs.

(2) it embeds: in 2 × 4mm²Template on write label (need to indicate simple sample ID and tissue block number). Label pendulum in embedding hole, after being placed in the roasting 15min of 60 DEG C of baking ovens, it is stand-by that 40 DEG C of incubator is placed in after taking-up.Then 40 It is embedded in DEG C insulating box.

(3) it polymerize: after sample has embedded, is put into baking oven and is polymerize, polymerization temperature and time is 37 DEG C, 12h；45℃, 12h；60℃,48h.

(4) it modifies tissue block: embedded block is clipped on sample collet, with single-edge blade accomplishing four at the sample of embedded block Face cone body.Afterwards under anatomical lens, smooth plane is accomplished at embedded block top with double-edged razor blade.

(5) it slice and dyeing: is sliced with Leica UC6 ultramicrotome, then with acetic acid uranium dye liquor and lead citrate Dye liquor is dyed.Finally, using observing and taking pictures under Hitachi's H-7500 transmission electron microscope.

(6) measurement of length of sarcomere is measured using Image pro plus software, and each sample is at least chosen under 7 visuals field 60, face muscle fibre.

1.3.6iTRAQ experiment flow

1.3.6.1 protein extraction

(1) appropriate amount of sample is weighed into 1.5ml centrifuge tube；

(2) a 5mm magnetic bead and appropriate Lysis Buffer 3 is added, adds the PMSF of final concentration of 1mM, 2mM respectively EDTA, stand 5 minutes after vortex oscillation, add the DTT of final concentration 10mM；

(3) 2 minutes (power=50HZ, Time=120S) is shaken with tissue grinder instrument；

(4) (4) 25,000g × 4 are centrifuged 20 minutes, take supernatant；

(5) plus final concentration 10mM DTT, 56 DEG C water-bath 1 hour；

(6) darkroom final concentration 55mM IAM is added after restoring to room temperature and stands 45 minutes；

(7) 4 times of volume cold acetones, -20 DEG C of standing 2h are added；

(8) it repeats step (7) two to arrive three times, until supernatant is colourless；

(9) 25,000g × 4 are centrifuged 20 minutes, abandon supernatant；

(10) a 5mm magnetic bead and appropriate Lysis Buffer 3 is added in precipitating；

(11) 2 minutes (power=50HZ, Time=120S) is shaken with tissue grinder instrument；

(12) centrifugation of 25,000g × 4 takes supernatant for quantitative after twenty minutes.

1.3.6.2 protein extraction Quality Control

(1) Bradford is quantitative

Standard protein (0.2 μ g/ μ l BSA) 0,2,4,6,8,10,12 is sequentially added in 96 hole elisa Plates A1 to the position A10, 14,16,18 μ l sequentially add 20,18,16,14,12,10,8,6,4,2 μ l of pure water later, and Coomassie brilliant blue is added in each hole later 180 μ l of G-250 paced work liquid.OD595 is measured with microplate reader, makes linear standard curve according to OD595 and protein concentration.It is dilute Testing protein solution several times are released, 180 μ l paced work liquid are added in 20 μ l protein solutions, read OD595.Establishing criteria Curve and sample OD595 calculate sample protein concentration.

(2)SDS-PAGE

Every sample takes 30 μ g protein solutions that 95 heating 5 minutes after appropriate loading buffer is mixed, 25,000g centrifugations are added 5 minutes, supernatant is taken to click and enter in the loading wells of 12%SDS polyacrylamide gel.120V constant pressure electrophoresis 120 minutes；Electrophoresis terminates Afterwards, appropriate destainer (40% ethyl alcohol, 10% acetic acid) is added later and is placed in shaking table destainer 3~5 for coomassie brilliant blue staining 2 hours It is secondary, 30 minutes every time.

1.3.6.3 proteolysis

(1) each sample takes 100 μ g protein solutions；

(2) in albumen: 2.5 μ g of Trypsin enzyme, 37 enzymatic hydrolysis 4 hours are added in enzyme=40:1 ratio；

(3) it is primary to add Trypsin again according to the above ratio, 37 continue enzymatic hydrolysis 8 hours；

(4) peptide fragment digested carries out desalination using Strata X column, and vacuum is drained.

1.3.6.4 peptide segment mark

(1) according to sample size, a certain amount of iTRAQ tagging reagents are taken out；

(2) candidate agent restores to room temperature, and 50 μ l isopropanols are added in every pipe reagent, low-speed centrifugal after the concussion that is vortexed；

(3) peptide fragment sample is dissolved with 0.5M TEAB, and be added in corresponding ITRAQ tagging reagents.Different sample peptide fragment choosings With different iTRAQ labels；

(4) static 2 hours of room temperature.

1.3.6.5 peptides separation

Using Shimadzu LC-20AB liquid phase systems, splitter is that 4.6 × 250mm of 5um Gemini C18 column carries out sample Liquid phase separation.The peptide fragment sample drained and sample introduction are redissolved with 2mL mobile phase A (5%ACN pH9.8), with 1mL/ minutes flow velocitys Gradient elution: 5% Mobile phase B (95%ACN, pH9.8) 10 minutes, 5% to 35% Mobile phase B 40 minutes, 35% to 95% stream Dynamic phase B 1 minute, Mobile phase B continued 3 minutes, and 5% Mobile phase B balances 10 minutes.Eluting peak and every is monitored under 214nm wavelength Minute collects a component, merges sample in conjunction with chromatography eluant peak figure and obtains 20 components, then freezing is drained.

1.3.6.6 efficient liquid phase

The peptide fragment sample mobile phase A (2%ACN, 0.1%FA) drained is redissolved, 20,000g centrifugations after ten minutes, take Supernatant sample introduction.It is separated by Thermo company UltiMate 3000UHPLC.Sample initially enters trap column and is enriched with and removes Salt is then connected with self-chambering C18 column (75um internal diameter, 3um column material > partial size, 25cm column length), with 300nl/min flow velocity by such as Lower effective gradient is separated: 0-5min, 5% Mobile phase B (98%ACN, 0.1%FA)；5-45min, Mobile phase B is from 5% line Property rises to 25%；45-50min, Mobile phase B rise to 35% from 25%；50-52min, Mobile phase B rise to 80% from 35%；52- 54min, 80% Mobile phase B；54-60min, 5% Mobile phase B.Nanoliter liquid phase separation end is directly connected to mass spectrograph.

1.3.6.7 Mass Spectrometer Method

Tandem mass spectrometer Q-Exactive HF is entered after the peptide fragment of liquid phase separation is by nanoESI source ion X (ThermoFisher Scientific, San Jose, CA) carries out DDA (data-dependent acquisition) mode Detection.Major parameter setting: ion source voltage is set as 2kV；350~1500m/z of first mass spectrometric scanning range；Resolution ratio setting It is 60,000；Second order ms starting m/z is fixed as 100；Resolution ratio 15,000.The parent ion screening conditions of second level fragmentation are as follows: electricity Lotus 2+ to 5+, peak intensity are more than that 10,000 intensity comes preceding 20 parent ion.Ion fragmentation mode is HCD, and fragment ion exists It is detected in Orbitrap.The dynamic exclusion time is set as 30s.AGC setting are as follows: level-one 3E6, second level 1E5.Mass spectrum terminates, Initial data is obtained, data processing and bioinformatic analysis are carried out.

1.3.6.8 protein identification

Mascot is the important identification software of proteomics field, uses extensive, software version used in the project For Mascot2.3.02.Raw mass spectrum data are changed by thermo scientific tool Proteome Discoverer MGF format, the MGF file to take a turn for the better later are searched for by identification software Mascot and the protein sequence database chosen comparison To final Identification of Fusion Protein result.Finally selected credible albumen must include at least one believable specific (Unique) peptide Section, specific search parameter are as follows:

Table 1-1 Mascot search parameter

1.3.6.9 protein iTRAQ is quantitative

The quantitative IQuant software using Hua Da independent research of ITRAQ data, the software incorporate Mascot Percolator algorithm, which automatically gives a mark again to database search result using machine learning algorithm, to mention The identification rate of high result.The filtering (FDR≤0.01 PSM-level) of 1%FDR is carried out in spectrogram/peptide fragment level first, thus Obtain spectrogram and the peptide fragment list of conspicuousness identification.Then it is based on " law of parsimony " (The parsimony principle), benefit Albumen assembling is carried out with peptide fragment, and generates a series of protein groups.In order to control the false positive rate of albumen, process can also be in albumen (FDR≤0.01 Protein-level) is filtered again with FDR1% in level, using strategy is Picked protein FDR.The workflow of IQuant is main including the following steps: protein filtering, reporter group label purity Correction, quantitative values normalization, missing values completion, protein quantification value calculate, and statistical check analysis, final result is shown.

Main IQuant quantitative parameter is contained in following table:

Table 1-2 IQuant quantitative parameter

1.3.6.10 differential protein GO enrichment analysis

In the GO enrichment analysis of differential protein, by significant difference albumen and as all identification albumen phases of background Than examining the GO entry for finding out significant enrichment using hypergeometry；The Pathway enrichment analysis principle of differential protein is similar to this. The formula that hypergeometry is examined is as follows:

In above formula, N indicates all numbers for identifying and capable of being matched to GO entry in albumen；N is indicated in significant difference albumen The number of GO entry can be matched to；M indicates all numbers for identifying and capable of being matched to some GO entry in albumen；M indicates significant The number of some GO entry can be matched in differential protein.If the Pvalue value that hypergeometry is examined represents difference less than 0.05 Albumen is in the GO entry significant enrichment.

1.4 data processings and statistical analysis

(1) for statistical analysis to test data using SPSS 20.0 and 2010 software of Microsoft Excel, as a result It is indicated with mean+SD；Duncan ' s analysis is carried out to indices, is carried out using SPSS Pearson came (Pearson) method Correlation analysis.

(2) mass spectrometric data converts software: ProteoWlzard

(3) protein compares software: Mascot2.3.02

(4) Pathway analytical database: KEGG Pathway Database

(5) GO analysis tool: AmiGO

(6)NCBInr

(7)UniProt

2, result and analysis

The influence of 2.1 suspending ways and maturation time to ox back longissimus pH value

As shown in table 2-1, after government official in 1h, Jingbi fetters hanging technology and preceding common heel string hanging technological disparity is significant, May during binding having time delay, cause pH significant difference.Maturation to 72h, 2 kinds of suspending ways carry on the back longest to beef Flesh pH value has no significant effect.

The pH value of beef longissimus dorsi muscle under table 2-1 difference suspending way and maturation time

Note: A, B: indicate different maturation time processing differences up to the level of signifiance (P < 0.05)；A, b: different hanging sides are indicated Formula processing difference reaches the level of signifiance (P < 0.05), and following table is identical.

The influence of 2.2 suspending ways and maturation time to ox back longissimus shear force value

(in figure, * indicates that two kinds of suspending ways are significant in 0.05 level difference of P <) as shown in Figure 1, after Xinjiang rivers government official In 1h, although significant difference (P > 0.05) is not present in 2 kinds of suspending way shearing forces, pass through the beef of Jingbi constraint hanging Sample shear force value be lower than common heel string hanging beef sample shear force value 1.05kg, maturation time arrive 72h when, 2 kinds hang There are significant difference (P < 0.05) for mode shearing force, after this illustrates that heel string hanging-Jingbi constraint mode can significantly improve government official Tenderness of beef utilizing in 72h is a kind of beef fast-ripenin technology.

The influence of 2.3 suspending ways and maturation time to ox back longissimus cooking loss

As shown in table 2-2, suspending way and maturation time have no significant effect (P > to beef longissimus dorsi muscle cooking loss 0.05)。

Table 2-2 difference suspending way and maturation time beef longissimus dorsi muscle cooking loss

2.4 suspending ways and maturation time change MFI in ox back longissimus

Myofibril Fragmentation is the immediate cause for causing muscle self-dissolving and meat to tenderize at the phenomenon that small fragment, so MFI It is considered as the important indicator for measuring meat tenderness degree.MFI can reflect muscle fibril and the complete journey of skelemin inside myocyte Degree, MFI is bigger, and muscle fibril internal structure integrality is bigger by the degree destroyed, and muscle maturity is higher.Such as table 2-3 institute Showing, the MFI value of Jingbi constraint hanging and the MFI value difference of common heel string hanging are different not significant (P > 0.05) in 1h after government official, and after killing The MFI value of 72h Jingbi constraint hanging and the MFI value significant difference (P < 0.05) of common heel string hanging, illustrate Jingbi constraint hanging The tenderization for the ox back longissimus that mode accelerates.The MFI value difference of 0d and 72h is anisotropic significant, illustrates the extension with maturation time, Myofibrillar lysis degree becomes larger.

Table 2-3 difference suspending way and maturation time beef longissimus dorsi muscle muscle fibril snip to change exponent are than variation

Muscle fibre Change of Ultrastructure in 2.5 hanging maturations

Ultra microstructure can directly observe kill after hang maturation in muscle fibril metamorphosis.After government official when 1h, neck Arm constraint hanging processing group and common heel string hanging processing group muscle fibril are clear in structure as it can be seen that muscle fibril is closely coupled, bright Band, blanking bar, M line, Z-line and the area H are complete, do not find phenomenon of rupture.After government official when 72h, common heel string hanging processing group appearance is not advised Then deformation starts the phenomenon that several connected muscle segments are degraded occur, and Z-line degradation is more obvious, and tension belt occurs in partial region With spasm band.The muscle segment gap of the Jingbi constraint hanging processing group of 72h obviously broadens after government official, and muscle fibril occurrence of large-area is broken Bad, dissolution, muscle fibril structure are seriously damaged, as shown in Figure 2.

The extraction and quality inspection of 2.6 sample protein matter

2.6.1 sample protein matter Concentration Testing result

Protein concentration standard curve is as shown in Figure 3.

Sample protein matter concentration is as shown in Table 2-4.

Table 2-4 sample protein matter concentration

SDS-PAGE electrophoresis detection result is as shown in Figure 4, wherein the concentration of separation gel is 12%, Marker applied sample amount 10 Microgram, whole applied sample amounts are 20 micrograms.

2.6.2 data quality accessment

Quantified in project in protein iTRAQ, share 4 groups of Bovine samples, be respectively as follows: BZC1, BZC2, BZC3, BZC4 has carried out 1 repetition altogether and has tested, and shares 892027 second level spectrograms and generates lower machine.Under " 1%FDR " filter criteria, one It shares 15309 peptide fragments and 2608 albumen is identified, as shown in table 2-5.

Table 2-5 protein identification data

2.6.3 the quantitative result of protein

2.6.3.1 comparison among groups analyze result

In the present invention, BZC1/BZC2, BZC3/BZC4, BZC1/BZC3, BZC2/BZC4 are arranged to comparative group.Single is real The significant difference albumen tested is with change>1.2 Fold and Q-value<0.05 two conditional filtering.Number is tested to being repeated several times For, final differential protein needs at least to be defined as differential protein in 1 repeated data.BZC1 is the common heel string of 1h after killing Processing group is hung, BZC2 is 1h Jingbi constraint hanging processing group after killing, and BZC3 is the common heel string hanging processing group of 72h after killing, BZC4 is 72h Jingbi constraint hanging processing group after killing.As shown in table 2-6, common heel string hanging processing group and Jingbi constraint hanging Processing group 1h upregulated protein 3 after government official, down-regulation protein 17；72h upregulated protein 11 after government official, down-regulation protein 1.

The significant difference protein profile that different suspending ways and maturation time compares is as shown in Figure 5.

Comparison result under table 2-6 difference suspending way and maturation time

2.6.4 albumen annotation analysis result

2.6.4.1 albumen KOG annotation analysis

Differential protein KOG annotation is that the entry for annotating each comparative group differential protein individually extracts, and draws column Shape figure is shown, can better understand the corresponding function classification of differential protein, as shown in Figure 6.

2.6.4.2 Protein G O annotation analysis

After all protein identified and NR database compare, corresponding GO functional information is obtained, GO function is divided into carefully Born of the same parents' component (Celluar Component), molecular function (Molecular Funtion), bioprocess (Biological Process) integration is carried out according to the GO functional information in comparison to summarize.As seen from Figure 7, all protein G O identified Annotation is mainly occupied at two aspects of bioprocess (Biological Process) and cellular component (Celluar Component) It is more.

2.6.4.3 albumen Pathway is annotated

As shown in figure 8, X-axis represents albumen annotation number, Y-axis represents KEGG function classification.KEGG metabolic pathway is divided into 7 A branch: cell processes (Cellular Processes), environmental information handle (Environmental Information Processing), hereditary information handles (Genetic Information Processing), human diseases (Human Diseasea) (only restraint object), metabolism (Metabolism), organic system (Organismal Systems), drug development (Drug Development)。

2.6.5 differentially expressed protein access is enriched with

2.6.5.1 differential protein GO is analyzed

As shown in Figure 9 and Figure 10, by Jingbi constraint hanging and the hanging of common heel string as the differential expression egg compared It is white to carry out the enrichment analysis of GO access respectively, access, two comparative groups are enriched with 0.05 threshold value standard screening conspicuousness of q-value < (the common heel string of 1h is hung in the constraint hanging of 1h Jingbi, and the common heel string of 72h is hung in the constraint hanging of 72h Jingbi) conspicuousness is enriched with GO Access is respectively as follows: 138 and 94, wherein significant access has: fibrinogen complex (fibrinogen complex), G protein coupled receptor combines (G-protein coupled receptor binding), peptidase inhibitors activity (peptidase inhibitor activity), peptase regulator active (peptidase regulator activity), Phosphatide combines (phospholipid binding), platelet activation (platelet activation), steroid metabolism process (steroid metabolic process), cholesterol metabolic process (cholesterol metabolic process) swash Plain metabolic process (hormone metabolic process), regulatory protein matter phosphorylation (regulation of protein Phosphorylation), positive regulator (the positive regulation of protein of protein phosphorylation Phosphorylation), regulatory protein matter metabolic process (regulation of protein metabolic process), The positive adjusting (positive regulation of phosphate metabolic process) of phosphate metabolism process, Positive adjusting (the positive regulation of protein metabolic process), enzyme of protein metabolism process Join receptor protein signal path (enzyme linked receptor protein signaling pathway), transmembrane receptor Protein Serine/threonine kinase signal path (transmembrane receptor protein serine/threonine kinase)。

2.6.5.2 differential protein Pathway access is enriched with

The significant difference albumen that different suspending ways are relatively obtained obtains each ratio in KEGG Pathway database Compared with group, in database, the Pathway access of each comparative group is obtained, with the method for GO access enrichment, with 0.05 threshold of p-value < It is worth the Pathway access of standard screening conspicuousness enrichment.As is illustrated by figs. 11 and 12, the Pathway access of two comparative groups point It is not: 47 and 42.Pathway significant enrichment is respectively then: 9 and 18.Wherein significant access has: D-Gln (Vitamin is digested and assimilated with D-Glu metabolism (D-Glutamine and D-glutamate metabolism), vitamin Digestion and absorption), fat digestion absorbs (Fat digestion and absorption), protein and disappears Change and absorbs (Protein digestion and absorption), relaxain signal path (Relaxin signaling Pathway), platelet activation (Platelet activation), alanine, aspartic acid and glutamic acid metabolism (Alanine, Aspartate and glutamate metabolism), ECM- acceptor interaction (ECM-receptor Interaction), Arginine biosynthesis (Arginine biosynthesis) etc..

By above-mentioned experiment it follows that

(1) suspending way is fettered by Jingbi, the shear force value of ox back longissimus 1h and 72h after government official are substantially lower than The muscle fibril snip to change exponent numerical value of the shearing force of common heel string suspending way, Jingbi constraint suspending way when 72h is significant Higher than the numerical value of common heel string suspending way, illustrate the ox back longissimus that heel string hanging-Jingbi constraint suspending way accelerates Tenderization.

(2) by being hung after Ultrastructural observation to government official in maturation in muscle fibril, heel string hanging-Jingbi constraint The length of sarcomere of the more common heel string suspending way processing of the length of sarcomere of suspending way processing is longer, heel string hanging-Jingbi constraint The degradation situation of hanging processing group becomes apparent.

(3) the brown ox back longissimus of the different suspending ways of 0h and 72h is identified by iTRAQ protein group quantitative technique Totally 2608, protein, the differentially expressed protein of BZC1VSBZC2, BZC3VSBZC4 comparison among groups has 20,12 respectively. The protein that identifies carry out functional annotation be mostly cell Proliferation, metabolic activity, in terms of；To comparison among groups Differentially expressed protein carry out the functional annotation overwhelming majority be all energy conversion, biosynthesis, signal transduction mechanism etc..All mirror Surely GO, Pathway access of the protein and comparison among groups differential expression protein that arrive are enriched with, wherein having many accesses and originally grinding It is related to study carefully main meat tenderness degree.

Claims (9)

1. a kind of method for improving beef quality after beef cattle kills, includes the following steps:

Keep beef mature by the way of heel string hanging-Jingbi constraint, and then improves beef quality；

The heel string hanging-Jingbi constraint mode refers to hanging beef side body in such a way that heel string is hung, then preceding Apply radial pull between arm distal end and ox neck.

2. according to the method described in claim 1, it is characterized by: the size of the radial pull is 10~50KG.

3. method according to claim 1 or 2, it is characterised in that: the beef quality refers to the tenderness of beef.

4. method according to any one of claim 1-3, it is characterised in that: the time of the hanging is 1h to 21 days.

5. heel string hanging-Jingbi is strapped in the application improved after beef cattle kills in beef quality；

The heel string hanging-Jingbi constraint mode refers to hanging beef side body in such a way that heel string is hung, then preceding Apply radial pull between arm distal end and ox neck.

6. application according to claim 5, it is characterised in that: the size of the radial pull is 10~50KG.

7. application according to claim 5 or 6, it is characterised in that: the beef quality refers to the tenderness of beef.

8. the application according to any one of claim 5-7, it is characterised in that: time 1h to 21 days of the hanging.

9. the application according to any one of claim 5-8, it is characterised in that: the application be presented as it is following 1) or 2):

1) shearing force of beef is reduced；

2) the muscle fibril snip to change exponent of beef is improved.