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A novel method for beef bone protein extraction by lipase-pretreatment and its application in the Maillard reaction
Accepted Manuscript A novel method for beef bone protein extraction by lipase-pretreatment and its application in the Maillard reaction Shiqing Song, Sisi Li, Li Fan, Khizar Hayat, Zuobing Xiao, Lihua Chen, Qi Tang PII: DOI: Reference:
S0308-8146(16)30434-4 http://dx.doi.org/10.1016/j.foodchem.2016.03.062 FOCH 18946
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
24 January 2016 13 March 2016 18 March 2016
Please cite this article as: Song, S., Li, S., Fan, L., Hayat, K., Xiao, Z., Chen, L., Tang, Q., A novel method for beef bone protein extraction by lipase-pretreatment and its application in the Maillard reaction, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.03.062
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A novel method for beef bone protein extraction by lipase-pretreatment and its application in the Maillard reaction Running title: Lipase-pretreatment extraction of protein from beef bone Shiqing Songa, Sisi Lia, Li Fana, Khizar Hayatb, Zuobing Xiao *,a, Lihua Chen a, Qi Tanga a
School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai,
201418, PR China b
Department of Food Science and Nutrition, College of Food and Agricultural Sciences,
King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia *
Corresponding author: +86-21-60873150; Fax: +86-21-60873424
E-mail: [email protected]
1
ABSTRACT: Five beef bone hydrolysates were obtained by different enzyme treatment schemes, including papain (M), combination of porcine pancreatic lipase and papain (Z+M, combination of lipase and papain (Y+M), Protamex (F), combination of porcine pancreatic lipase and Protamex(Z+F). The degree of hydrolysis (DH), free amino acids and molecular weight distribution of these hydrolysates were evaluated. To further explore the differences between these five hydrolysates, Maillard reaction
products
(MRPs)
were
prepared
using
a
xylose/cysteine/hydrolysate model. It was found that the DH, content of low molecular weight peptides and amino acids of hydrolysates increased significantly after lipase pre-treatment. GC-MS showed that the total content of furans, pyrroles and thioethers in MRPs Y+M increased by 78.0% compared with MRPs M, while in MRPs Z+F, pyrazines increased by 44.1% compared with MRPs F. Examining the sensory characteristics of the MRPs, the MRP from the hydrolysate of Y+M had the best mouthful, umami and meaty characteristics. The correlation analysis further confirmed that an appropriate lipase pre-treatment could improve the flavour of MRPs. Keywords:
Bone
hydrolysates;
lipase;
Maillard
reaction
product;
Descriptive sensory analysis; GC-MS; partial least squares regression. Chemical compounds studied in this article: 2
Xylose (PubChem CID: 135191); Cysteine (PubChem CID: 5862); Dimethyl disulfide (PubChem CID: 12232); Dimethyl trisulfide (PubChem CID: 19310); 2-Pentylfuran (PubChem CID: 19602); Hexanal (PubChem CID: 6184)
3
1. Introduction Approximately 48.28 million cattle were slaughtered in China in 2013 (State Statistical Bureau, 2014). Most of this beef is deboned in plant and the meat is sold as packaged beef. During this process, as much as 6‒12% (based on carcase weight) of bone was left. This could result in approximately 4.43 million tonnes of bone (almost no meat attached to the bone) disposed of or sold at a lower price as inedible by-products (Wang et al., 2015). Beef bone contains a notable amount of muscle, connective tissue and fat, and therefore represents a valuable source of proteins, containing about 47% moisture, 21% protein (collagen), 15% fat, and 15% ash. Non-utilisation or underutilisation of animal by-products not only leads to loss of potential revenues but also leads to a higher cost of disposal of these products. For that reason, industries have begun to develop various technologies to make use of this waste, mainly in the form of value-added products, at the same time reducing the cost derived from its disposal. Continuous efforts have been made to improve the functional and nutritional value of bone. Boles et al. (2000) used different solutions (4% sodium chloride, 4% sodium chloride with either 0.3 M sodium tripolyphosphate, tetrasodium pyrophosphate or 0.05 M NaOH to effectively extract proteins from beef bones. These proteins could be used to manufacture finely comminuted sausage products with similar texture to 4
sausages made with commercially available proteins. Nikolaev et al. (2008) stated that a functional meat protein could be obtained by the fermentation of meat-bone broiler residues. Recently, enzymatic hydrolysis was employed to extract proteins and produce peptides (Morimura et al., 2002), which formed an effective way to recover proteins from the by-products of animal processing. Unlike acidic or alkaline hydrolysis, enzymatic proteolysis is mild and controllable, which helps to improve the quality and functional properties of protein (Kristinsson & Rasco, 2000). Linder et al. (1995) reported that the utilisation of bone mainly focused on the enzymatic extraction of nutrients. After hydrolysis, bone could be developed into value-added products. Linder et al. (1997) also described that the enzymatic hydrolysate of veal bone contained a large amount of glycine and proline, whose nutritional value was much higher than the hydrolysate treated by acid or alkali. It was found that the hydrolysates were useful for soups, sauces and gravies. Dong et al. (2014) used hot-pressure combined with enzymolysis to extract protein from chicken bone; these hydrolysates demonstrated a new kind of potential suitable nutritional supplement in various foods. Other researchers also found that the hydrolysed protein was an important flavouring agent (Lieske & Konrad, 1994; Lafarga & Hayes, 2014; Zhan et al., 2013), which could give Maillard reaction products (MRPs) with lifelike meat flavour (Pommer, 1995). 5
The water-soluble meat flavour precursors consist of free amino acids, peptides, and reducing sugars (Khan, Jo, & Tariq, 2015). Madruga et al. (2010) reported that by controlling the degree of hydrolysis (DH), different constituents of these precursors could generate different flavours. In general, the lower the DH is, the fewer the precursors. It is well-known that beef bone is usually surrounded by adipose tissue, which may prevent the combination of protein and protease, leading to a low degree of hydrolysis. Linder et al. (1997) hydrolysed veal bone using Neutrase only, with an unsatisfactory DH. Therefore, it is necessary to develop an effective way for better utilisation of beef bone by-product. Since lipase could hydrolyse redundant adipose tissue during lean meat processing, this enzyme was chosen to pre-treat the beef bone. The objective of present study is to develop a new method for the preparation of protein from beef bone, and compare it with other methods. The study includes(A) analysis of the degree of hydrolysis, free amino acids and molecular weight of five bone hydrolysates hydrolysed by different treatment schemes, including papain, combination of porcine pancreatic lipase and papain, combination of lipase and papain, Protamex, combination of porcine pancreatic lipase and Protamex;
(B) Comparison of the sensory
characteristics and the volatile compounds of the MRPs prepared from the five hydrolysates. (C) Study of the relationship between free amino acids, 6
molecular weight distribution of hydrolysates and the sensory characteristics of MRPs. Through the above analyses, the influence of the lipase pre-treatment on beef bone hydrolysate and MRP was investigated. 2. Materials and methods 2.1. Chemicals and materials Beef bone was purchased from Shanghai Tesco Supermarket (Shanghai, China). Xylose and cysteine were purchased from Sigma China Co., Ltd. (Beijing, China). Papain (2000 U/mg) and Protamex (31.4 U/mg) were obtained from Novo Co., Ltd. (Novozyme Nordisk, Bagsvaerd, Denmark). Porcine pancreatic lipase (20 U/mg) was purchased from Shanghai Source Poly Biological Technology Co., Ltd. (Shanghai, China). Lipase (Yiming, 20 U/mg) was purchased from Yiming Biological Products Co., Ltd (Jiangsu, China). Formaldehyde and NaOH were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The other chemical reagents were purchased from National Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Preparation of bone hydrolysates hydrolysed by different enzymes Beef bones were first cleaned of meat, fat and bone marrow and heated for 4 h at 121 °C, 0.1MPa a in pressure vapour steriliser (Shanghai Shenan Medical Devices Co., Ltd., Shanghai, China). Then it was dried at 60 °C for 4 h before grinding into powder (80 mesh size) by high-speed grinding machine (Tianjin Instrument Co., Ltd., Tianjin, China). Bone powder was 7
mixed with deionised water in a certain proportion. Then the mixture was hydrolysed by different enzymes. The preparations of five different beef bone hydrolysates are listed in Table 1. All five hydrolysates were prepared at the optimal conditions of the enzymes. The composite enzymatic hydrolysates were hydrolysed by porcine pancreatic lipase or lipase for 3 h, followed by papain or Protamex for 3 h. After the enzyme deactivation at 90 ℃ for 10 min, these five hydrolysates (designated M, F, Z+M, Z+F, Y+M,) were centrifuged (Scientific Instrument Co., Ltd., Shanghai, China) at 4000 g for 20 min. The supernatants were kept at 4 ℃ until used. 2.3. Determination of DH DH of the hydrolysates was measured according to Song et al. (2013). 2.4. Free amino acid analysis A pre-treatment of the sample was needed before the amino acid analysis. For the determination of free amino acids, 5 mL sample were added to a volumetric flask (25 mL), and 5% trichloroacetic acid (TCA) was added to volume, to precipitate peptides or proteins (Song et al., 2013). The solution was filtered through Whatman filter paper No.4 after incubation for 2 h at room temperature. Then the filtrate was centrifuged (Scientific Instrument Co., Ltd., Shanghai, China) at 12300 g for 10 min and stored at 4 ℃. Amino acids in bone hydrolysates were analysed (Song et al., 2013; Liu et al., 2012). The sample (20 µL) was injected into an automated online 8
derivatisation system with and analyzed by an Agilent 1100 HPLC with UV detector operated at 338 nm/262 nm using a Hypersil ODS column（4.6 mm × 250 mm × 5 µm）, while the flow rate was 1 mL/ min. Temperature of the column was 40 ℃. The mobile phase was composed of 0.6 mM sodium acetate and 0.15 mM sodium acetate: acetonitrile: methanol, (1:2:2, v:v:v). Amino acidsstandards (Sigma Chemical Co., St. Louis, MO) were used for the determination of a calibration curve for calculation, and the identification and quantification were decided on the basis of the retention time and the peak area of standard compounds, respectively. 2.5. Molecular weight distribution analysis The molecular weight distribution of the bone hydrolysates was analysed by a Waters 600 liquid chromatography system (Waters Co., Milford, MA,). The system was equipped with a Waters 2487 UV detector and Empower work station on a 2000 (300 × 7.8 mm) SWXL TSK gel filtration column (Tosoh
Co.,
Tokyo,
Japan).
The
mobile
phase,
consisting
of
acetonitrile/water/trifluoroacetic acid (45:55:0.1, v:v:v), was delivered at a flow rate of 0.5 mL/ min. The column temperature was 30 ℃ and 10 µL sample were injected into the HPLC system. The molecular weight calibration curve was obtained using five standards from Sigma: cytochrome C (12500 Da), aprotinin (6500 Da), bacitracin (1450 Da), tetrapeptide GGYR (451 Da), and tripeptide GGG (189 Da). The chromatogram was 9
recorded using a UV detector at 220 nm. The data were analysed by gel permeation chromatography (GPC) software. 2.6. Preparation of MRPs The supernatants of hydrolysates were concentrated by a rotary evaporator till the solids content was 20%. Then cysteine (0.4 g) and xylose (0.64 g) were added into 20 mL concentrated supernatant. The mixture was adjusted to pH 7.0 with 6 M NaOH, then transferred into a reaction vessel and allowed to react in an oil bath with magnetic stirring (130 rpm) at 110 °C for 90 min. These five Maillard reaction products (marked as MRPs M, F, Z+M, Z+F, Y+M, respectively) were immediately cooled in ice-water and stored in a refrigerator (‒18 °C) for further analyses. Three control samples were prepared from (1) hydrolysate of beef bone, (2) cysteine and xylose, beef bone powder, and (3) cysteine and xylose, respectively. Their preparation conditions were treated as mentioned above, and they were named M, MRPs 1 and MRPs 2, respectively. 2.7. Sensory characteristics of MRPs To ascertain the sensory characteristics of these five Maillard reaction products, sensory evaluation was carried out according to the method of Schlichtherle-Cerny
and
Amadò
with
some
modifications
(Schlichtherle-Cerny & Amadò, 2002). The evaluation of the samples was done by a panel of five males and three females (23‒45 years old) from the 10
School of Perfume and Aroma Technology at Shanghai Institute of Technology, China. All panellists had numerous experiences in sensory evaluation of foods. The eight panellists had previously been trained for 3 h to define descriptive terms and to decide suitable reference solutions for the samples. The reference solutions were prepared as follows: 2.0 g Hershey’s syrup caramel (Hershey (Shanghai) Co., Ltd) in 40 mL water was labelled caramel-like, 3 mM MSG (Shanghai Totole Food Co., Ltd) was labelled umami, 15 mM lactic acid (Henan Jindan Technology Co., Ltd) was labelled sour, defatted brisket meat (0.5 kg, 2.5-cm thick, purchased from Wal-Mart supermarket) boiled in water (1 kg) for 2 h was labelled meat aroma; 2.0 mM caffeine (Illycaffe (Shanghai) Trading Co., Ltd) was labelled bitter, and 10 g bouillon cube (beef flavour consisting of MSG, yeast extract, and beef extract), dissolved in water was labelled mouthful. The evaluation was implemented using a 1‒10 interval scale (0 = none, 10 = extremely strong), where 5 points was the score of the reference solutions. The sensory evaluation took place in a sensory laboratory equipped to international standards. Maillard reaction products (60 mL) and 60 mL reference solutions were tasted at 22 ± 2 ℃ in separated sensory booths at the same time. 2.8. GC-MS analysis The volatile compounds were extracted by SPME with a 75 µm Carboxen-PDMS fibre (Supelco, Bellefonte, PA). Maillard reaction product 11
(10.0 g) was placed into a glass vial and 50 µL internal standard 1,2-dichlorobenzene in methanol (0.5 µg/µL) was added into the sample before trapping. The glass vial was sealed with a lid and placed in a water bath at 55 ℃ for 30 min with aging the SPME fibre in the headspace to allow the equilibration of the volatile compounds. Then the fibre was desorbed at 250 ℃ for 5 min. Separation of the volatile compounds was implemented on an HP-INNOWAX (60 m × 0.25 mm, 0.25 µm; J & W Scientific, Folsom, CA) capillary column. Helium was used as the carrier gas, flowing at 1.8 mL/min. The starting temperature of the chromatographic column was 50 ℃, held for 3 min, then increased up to 230 ℃ at 3 ℃ / min, and then held at this temperature for 7 min. The mass spectrometric detector was operated at an electron voltage of 70 eV and the temperature of the ion source was 230 ℃. Volatile compounds were identified by comparing the detector data (Kovats retention Indices, KI) of samples with authentic standards, the published literature, the NIST 11 and the Wiley 07 databases. The KI values were calculated relative to an n-alkanes series (C8‒C23) under the same condition of the samples. Approximate quantities of the volatile compounds were estimated by comparison of their peak areas with that of the internal standard, obtained from the total ion chromatograms, assuming that the relative response factor was 1 and the recovery ratio was 100%. The quantitative formula was as follows: 12
Wi = f ' *
Ai * ms /m As
where Ai is the peak area of compound i, As is the peak area of internal standard, ms is the mass of internal standard, m is the mass of sample, f’ is a relative correction factor, assumed to be 1, Wi is the concentration (µg/g) of compound i. 2.9. Statistical analysis Data from DH, free amino acids and sensory evaluation were subjected to analysis of variance by SPSS version 13.0 (SPSS Inc., Chicago, IL). Partial least squares regression (PLSR) was used to obtain the potential correlations among free amino acids, molecular weight distribution and sensory characteristics. All variables were centred and scaled to 1/SD so that each variable has a unit variance and zero mean before applying PLSR analysis, and to obtain an unbiased contribution of each variable to the criterion. All regression models were validated by full cross-validation. Multivariate analysis was performed with Unscrambler version 9.7 (CAMO ASA, Oslo, Norway). 3. Results and discussion 3.1. Effects of lipase pretreatment on DH of bone hydrolysates The protein hydrolysates of beef bone were prepared according to the method as mentioned above. The beef bone was hydrolysed by papain,
13
combination of porcine pancreatic lipase and papain, and combination of lipase and papain. The corresponding DH was 15.43 %, 23.17 % and 19.17 %, respectively (Table 1). DH values of these three bone hydrolysates were significantly different. It was found that DH of hydrolysates Z+M and Y+M were significantly higher than that of the hydrolysate which was treated with papain alone. Meanwhile, the porcine pancreatic lipase was more efficient in increasing the DH of the hydrolysate than lipase. In addition, another experiment using Protamex alone and combination of porcine pancreatic lipase and Protamex was carried out. The result indicated that DH increased greatly after the porcine pancreatic lipase pre-treatment of the samples (DH values of F and Z+F were 12.71% and 21.28%, respectively). From the above results, it is clear that the lipase pre-treatment exerted a considerable influence on the increase of DH. The possible reason was that after the lipase pre-treatment, more cleavage points were exposed, which made the interaction between proteins and enzymes easier; as a result the DH value increased. Moreover, compared with Z+F, the DH of Z+M was somewhat higher. 3.2. Analysis of free amino acids It is known that the composition and the content of the free amino acids have an important influence on the flavour properties of Maillard reaction 14
products directly and indirectly (Lorenzen, Davuluri, Adhikari, & Grün, 2005). As shown in Table 2, the lipase pre-treatment had an important influence on the content of free amino acids. The total free amino acids content of the samples hydrolysed by combination of lipase and protease significantly increased to that hydrolysed by protease alone. The total free amino acids of M, Z+M, Y+M, F and Z+F were 152 mg/g, 534 mg/g, 505 mg/g, 105 mg/g and 506 mg/g, respectively. It was obvious that Arg, Gly, Ala, Lys and Tyr were present at high proportion in all samples (Table 2). Griffith and Hammond (1989) reported that Lys could form alkylpyrazines and 2-acetyl-1-pyrroline through thermal reactions, while some potent flavour compounds are formed from cysteine and ribose through the Maillard reaction (Chen et al., 2000; Cerny, & Davidek, 2004; Yu et al., 2012). Meanwhile, enzymatic bone hydrolysates contain a larger portion of peptides than that of free amino acids, owing to the higher selectivity and specificity of enzymes. Some peptides could affect the flavour and aroma of the hydrolysate and the Maillard reaction products. It was reported that peptides of different structures and lengths could provide unique taste properties, such as umami, sour, bitter, salty and sweet (Spurvey et al., 1998). Val, Leu, Ile, Met, Phe, Ser, Arg and His are bitter amino acids; among these 8 bitter amino acids, Arg, His and Met are strongest in bitterness (Yang & Liu, 1983; Lan et al., 2010). Sample Z+F had a high content of all these 15
bitter amino acids. Umami taste was provided by Glu and hydrophilic amino acid residues (Arai et al., 1972; Arai et al., 1973). After the lipase pretreatment, the Glu decreased in Z+M, Y+M and Z+F, compared with M and F. Sour taste was closely related to umami. Kirimura et al. (1969) reported that Gly-Asp, Ala-Glu, Glu- Leu and other peptides had sour taste. 3.3. Analysis of molecular weight distribution The molecular weight distributions of five bone hydrolysates are shown in Table 3, which displays that these samples were composed of a series of low molecular weight polypeptides, especially peptides with molecular weight less than 1000 Da. As the DH increased, amino acids and some peptides less than 180 Da gradually increased. By contrast, the tendency of peptides above 500 Da was converse to that of those less than 180 Da. However, no apparent tendency was found in the peptides from 180 Da to 500 Da. Ogasawara et al. (2006a, 2006b) reported that the main compounds that provided special flavour were Maillard peptides whose molecular weight ranged from 1000 Da to 5000 Da. It was found that Maillard peptides decreased after the lipase pre-treatment. Nevertheless, by the cross linking of peptides below 1000 Da during the Maillard reaction, the content of high molecular weight peptides increased (Zamora & Hidalgo, 2005), particularly the Maillard peptides (Lan al., 2010). Because these peptides contribute to the mouthful flavour, the increase of these peptides could improve the 16
mouthful flavour and improve the thin taste of the product. 3.4. Sensory analysis of the MRPs The five Maillard reaction products were described as caramel-like, umami, sour, meaty, bitter and mouthful. An analysis of variance for MRPs was made, three replicates were applied to sensory data to assess the results. Significant differences among samples (p ≤ 0.05) for all attributes indicated that the samples had different aroma intensities. The mean intensity values of the 6 attributes and the results of Duncan’s multiple comparison tests are shown in Table 4. Comparing these five MRPs, MRPs F, Z+M, Z+F had a stronger caramel-like flavour, possibly because they were rich in furans (Van Boekel, 2006). Owing to the decrease of the bitter amino acids and the cross-linking of the peptides below 1000 Da (Lan et al., 2010), MRPs M, and F were more bitter than MRPs Z+M, Y+M and Z+F. Sour attribute increased after the lipase pre-treatment. MRPs
Y+M presented the highest
meaty, umami and mouthful attributes. Ogasawara et al. (2006b) reported that the cross-linking of peptides below 1000 Da could enhance mouthful and umami tastes. However, it was noted that MRPs Z+M and Z+F were much weaker in mouthful and umami than MRPs M and F. 3.5. GC-MS analysis of MRPs The volatile compounds of these five Maillard reaction products and three test samples (M, MRPs 1 and MRPs 2) were detected by GC-MS and 17
the analysis results are shown in Table 5. The lipase pre-treatment affected both the number of volatile compounds and the aroma intensity. A total of 64 compounds were detected and identified, including furans, pyrazines, thioethers, aldehydes and others that might have no contribution to the aroma characteristics of these eight samples. As shown in Table 5, few volatiles were detected in M, MRPs 1 and MRPs 2. M had the fewer kinds and amount of volatiles, than MRPs 1 and MRPs 2. More volatiles were detected in MRPs 2 than in MRPs 1. Furans and pyrazines were the dominant volatile compounds in the other five MRPs. After the lipase pretreatment, it was observed that the total content of furans, pyrroles and thioethers in MRPs Z+M and Y+M increased by 30.1% and 78.0%, pyrazines decreased by 10.5% and 38.7%, respectively, compared with MRPs M. In MRPs Z+F pyrazines increased by 44.1%, furans and thioethers decreased by 45.5% and 51.8%, respectively, compared with MRPs F. It was known that meaty flavour was associated with nitrogenous, sulphur-containing heterocyclic compounds and their derivatives (Song et al. 2013). Van Boekel (2006) reported that meaty flavour was mainly from sulphur compounds. Alkylpyrazines have also been detected in meat products (Mottram, 1998). Although sulfur volatiles have been demonstrated to be responsible for meat aroma, only dimethyl disulfide, dimethyl trisulfide and furfuryl methyl disulfide were detected in these samples. 18
MRPs F and MRPs Z+M had the most thioethers; however, the flavour of these two samples were not the best. These essential meaty flavour compounds were most abundant in MRPs F. However, Table 4 showed that MRPs Y+M had the highest mouthful, umami and meaty characteristics, while the overall flavour of MRPs F was not favoured. One reason might be that these aroma compounds were not contributing to the beef flavour. Another possible reason could result from the high content of furans, which possessed high caramel-like flavour, decreasing the meaty aroma. 3.6. Relationship between free amino acids, molecular weight of hydrolysates and sensory characteristics of MRPs To investigate which chemical parameters have a great effect on the flavour of the MRPs, ANOVA with PLSR was used to study the main scores accumulated from sensory evaluation and chemical parameters (free amino acids and molecular weights). The X matrix was designated to free amino acids and molecular weight; the Y matrix was designated to sensory attributes and the five MRPs (Fig. 1). The optimal number of components in the ANOVA- PLSR model presented was determined to be 3 Principal components PC1 versus PC2, PC1 versus PC3, and PC2 versus PC3 were explored, but PC1 versus PC3 is not presented here, as additional information was not obtained through this plot. Further PCs did not provide any predictive improvement on the Y matrix obtained. In this model session, 19
level effects were removed and the data were normalised through mean centring. The calibrated explained variances for these models were PC 1 = 59 %, PC 2 = 28 % and PC 3 = 10 %, respectively. As indicated from Fig. 1A, MRPs M and MRPs F were located on the left side, MRPs F correlated with peptides above 500 Da, and four amino acids Ala, Asp, Glu and Gly. MRPs M correlated with Met, Val, His, Phe, Leu, Ile and Arg. These amino acids are associated with bitter attributes to a great extent. Our results are consistent with the finding of Lan et al.(2010). MRPs Z+M, MRPs Y+M and MRPs Z+F were located on the right side and were significantly distinguishable from MRPs F and MRPs M. Thedifferent enzymatic treatments of beef bone had a significant influence on Maillard reaction products flavour. Further information can be obtained from Fig. 1B. Sample MRPs F is on the left side, which also correlated with amino acids Ser and Cys-S, sour and caramel-like aromas. MRPs Y+M and MRPs Z+M were located on the right side and they were associated with Lys and Arg. as well as with peptides of molecular weight less than 180 Da, umami, meaty, bitter and mouthful attributes. The sensory evaluation results also showed (Table 3) that MRPs Y+M had the highest score in umami, meaty and mouthful attributes. MRPs Z+F correlated with His, Phe, Val, Met, Leu, Ile and peptides from 180 Da to 500 Da. From the above results, it was observed that the aroma and flavour 20
of the MRPs could be improved by an appropriate pre-treatment of lipase. 4. Conclusions This study clearly revealed that lipase pre-treatment was efficient in increasing DH, the content of peptides below 1000 Da and free amino acids of bone hydrolysates. After the cross-linking of sugars and amino acids through the Maillard reaction, the aroma and flavour of Maillard reaction product from added bone hydrolysate by the pre-treatment of a combination of lipase and papain had the best taste with mouthful, umami and meaty characteristics. Further investigation by GC- MS showed that the volatile compounds increased, when compared with Maillard reaction products from bone hydrolysate prepared using papain alone. Meanwhile, PLSR analysis among molecular weight, free amino acids and sensory attributes of MRPs clearly showed that an appropriate pre-treatment of lipase could improve the flavour through thermal reactions. Hence, the protein hydrolysate of lipase and papain could be used to generate beef flavour. Acknowledgements The research was supported in part by National Natural Science Foundation of China (31201415), School project of Shanghai Institute of Technology (YJ2013-22) and Shanghai Engineering Technology Research Center of Fragrance and Flavour (12DZ2251400). References 21
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aromatic fingerprinting using HS-SPME-GC-MS and multivariate analysis. Journal of Food Science, 79, 284-294. Yang, B. G., & Liu, S. C. (1983). Studies on the bitterness of protein. Food Science, 12, 1-2. Yu, A. N., Tan, Z. W., & Wang, F. S. (2012). Mechanism of formation of sulphur aroma compounds from L-ascorbic acid and L-cysteine during the Maillard reaction. Food chemistry, 132,1316-1323. Zamora, R., & Hidalgo, F. J. (2005). Coordinate contribution of lipid oxidation and Maillard reaction to the nonenzymatic food browning. Critical Reviews in Food Science and Nutrition, 45,49-59. Zhan, P., Tian, H., Zhang, X., & Wang, L. (2013). Contribution to aroma characteristics of mutton process flavor from the enzymatic hydrolysate of sheep bone protein assessed by descriptive sensory analysis and gas chromatography olfactometry. Journal of Chromatography B, 921,1-8. Figure Captions Fig.1. The overview of the variation found in the mean data from the PLSR correlation loadings plot for five MRPs. The loadings of the X- and Y-variables for (A) PC 1 versus PC 2, (B) PC 2 versus PC 3 are presented. The model was derived from free amino acids and molecular weight of peptides of bone hydrolysates as the X matrix and sensory attributes of MRPs as Y matrix. Elipses represent r2 = 0.5 and 1.0. 27
Fig. 1
29
Table 1 Preparation of five different beef bone hydrolysates hydrolysates
M
Z＋M
Y＋M
optimum conditions of lipase pretreatment
—
porcine pancreatic lipase temperature: 35 ℃; pH = 7.0; time: 3 h; enzyme/substrate ratio 1.5% (w/w) lipase temperature: 35 ℃; pH = 7.5; time: 3 h; enzyme/substrate ratio 1.5% (w/w)
F
—
Z＋F
porcine pancreatic lipase temperature: 35 ℃; pH = 7.0; time: 3 h; enzyme/substrate ratio 1.5% (w/w)
optimum conditions of protein treatment papain temperature: 60 ℃; pH 3 h; enzyme/substrate (w/w) papain temperature: 60 ℃; pH 3 h; enzyme/substrate (w/w) papain temperature: 60 ℃; pH 3 h; enzyme/substrate (w/w)
degree of hydrolysis (%)
= 6.0; time: ratio: 1.0%
15.43b ± 0.05
= 6.0; time: ratio: 1.0%
23.17e ± 0.11
= 6.0; time: ratio: 1.0%
19.17c ± 0.12
Protamex Temperature: 40 ℃; pH= 6.5; Time: 3 h; Enzyme/substrate ratio: 1.0% (w/w) Protamex Temperature: 40 ℃; pH= 6.5; Time: 3 h; Enzyme/substrate ratio: 1.0% (w/w)
12.71a ± 0.01
21.28d ± 0.16
Values bearing different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
30
Table 2 Free amino acid analysis of the five bone hydrolysates (mean ± SDx ) concentration (mg/g) amino acid
M a
Asp Glu Ser His Gly Thr Arg Ala Tyr Cys-S Val Met Phe Ile Leu Lys Pro x
8.56 0.01 14.68e 0.03 1.71a 0.01 3.83b 0.02 31.91b 0.01 2.53a 0.01 34.76b 0.04 13.45b 0.02 1.53a 0.01 1.69a 0.01 6.10b 0.01 5.42b 0.01 8.68b 0.02 6.38b 0.01 5.78b 0.05 4.59b 0.03 0.35b 0.01
Z+M d
± 19.51 0.01 ± 6.08b 0.02 ± 12.57e 0.14 ± 7.18c 0.08 ± 88.26d 1.13 ± 22.52d 0.01 ± 83.47d 1.47 ± 40.40d 0.56 ± 63.37e 0.32 ± 19.34d 0.19 ± 14.06d 0.01 ± 13.64c 0.01 ± 21.87c 0.45 ± 14.30c 0.11 ± 13.09c 0.01 ± 83.78e 1.19 ± 10.23e 0.01
Y+M c
± 17.42 0.01 ± 5.51a 0.01 ± 11.95d 0.03 ± 9.08d 0.02 ± 76.02c 1.16 ± 22.92d 0.71 ± 82.56c 1.55 ± 36.17c 0.71 ± 55.03d 0.04 ± 17.03c 0.02 ± 13.79c 0.01 ± 16.17d 0.17 ± 26.65d 0.25 ± 16.58d 0.01 ± 14.70d 0.02 ± 77.48d 0.51 ± 5.63d 0.05
F b
± 9.11 0.01 ± 10.78d 0.02 ± 2.28b 0.01 ± 1.60a 0.01 ± 23.31a 0.17 ± 3.12b 0.01 ± 16.68a 0.24 ± 9.93a 0.01 ± 6.32b 0.01 ± 4.20b 0.01 ± 2.80a 0.02 ± 3.27a 0.01 ± 4.62a 0.01 ± 3.27a 0.11 ± 3.01a 0.01 ± 0.57a 0.01 ± 0.17a 0.01
Z+F
± 19.67d 0.98 ± 6.68c 0.02 ± 11.18c 0.17 ± 12.31e 0.05 ± 88.49d 1.22 ± 18.64c 0.05 ± 85.04e 0.44 ± 42.10e 0.17 ± 48.81c 0.15 ± 25.33e 0.02 ± 18.80e 0.01 ± 16.55e 0.01 ± 28.03e 0.02 ± 17.69e 0.02 ± 17.16e 0.17 ± 46.14c 0.49 ± 3.29c 0.24
Results were expressed as mean value ± standard deviation (n = 3).
Values bearing different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
31
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Table 3 Changes of molecular weight distribution (percent of total area) in five bone hydrolysates Samplesx MW(Da) M 0.03b ± >5000
Y＋M
F
Z＋F
0.02b ±
0.13a ±
0.04b ±
0.01
0.01
0.01
Z＋M c
0.00 ± 0.00 0.01 0.21c ±
0.12c ±
0.17c ±
0.71a ±
0.50b ±
0.06
0.01
0.03
0.03
0.01
1.18c ±
0.71d ±
0.83d ±
1.76a ±
1.38b ±
0.09
0.06
0.05
0.02
0.01
8.11b ±
4.46e ±
5.00d ±
10.10a ±
6.61c ±
0.08
0.01
0.05
0.02
0.02
25.85b ±
16.58d ±
16.86c ±
28.54a ±
16.38e ±
0.09
0.02
0.04
0.01
0.01
52.75c ±
58.58b ±
58.90a ±
49.27d ±
58.98a ±
0.07
0.03
0.01
0.01
0.05
12.61d ±
19.57a ±
18.28b ±
9.55e ±
16.12c ±
0.35
0.01
0.01
0.06
0.06
3000‒5000
2000‒3000
1000‒2000
500‒1000
180‒500
<180 x
Peptides in bone hydrolysates as percent of total area (%).
Values bearing different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
32
Table 4 The mean scores of the 5 attributes for the 5 MRPs in descriptive sensory evaluation sample
mean score caramel-like
umami
sour
meaty
bitter
mouthful
MRPs – Mx
5.67b
6.19d
3.78a
6.21d
7.96e
6.34d
MRPs - Z+M
7.32d
4.22a
7.92d
3.87a
6.65d
4.94b
MRPs - Y+M
4.80a
7.75e
6.88b
8.14e
4.40a
8.28e
MRPs - F
6.67c
6.08c
7.63c
5.86c
6.24c
5.64c
MRPs - Z＋F
8.12e
4.87b
8.26e
4.32b
5.32b
4.32a
x
MRPs - M/MRPs - Z+M/MRPs - Y+M/MRPs - F/MRPs - Z+F represented the Maillard reaction
products from M/Z+M/Y+M/F/Z+F, respectively. Mean scores for each attribute with different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
33
Table 5 Volatile compounds of eight MRPs analysed by GC-MS Compounds
KIx
IDy
Concentration (µg/100 g)*10 ± SDz M
MRPs - 1
MRPs - 2
MRPs - M
MRPs - Z+M
MRPs - Y+M
MRPs - F
MRPs - Z+F
furans 2-methylfuran
883
B
0.00g ± 0.00 5.48f ± 0.06 7.74e ± 0.06 7.97e ± 0.23
27.23d ± 0.09
33.99b ± 0.13 41.65a ± 0.61 33.66c ± 0.43
2-pentylfuran
1224
B
0.00f ± 0.00 0.00f ± 0.00 0.95c ± 0.02 0.53e ± 0.01
1.19b ± 0.02
0.71d ± 0.05
2.19a ± 0.16
0.00f ± 0.00
2-acetylfuran
1502
B
0.00g ± 0.00 1.07f ± 0.06 1.31e ± 0.01 1.58d ± 0.11
2.02c ± 0.03
2.32b ± 0.01
2.78a ± 0.11
2.71a ± 0.03
2-(methoxymethyl)furan
1232
A
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.17a ± 0.01
0.14b ± 0.01
0.00c ± 0.00
furfural
1443
A,B 0.00h ± 0.00 5.55f ± 0.14 1.44g ± 0.01 40.15d ± 0.17
31.64e ± 0.10
53.64b ± 0.06 97.58a ± 0.03 42.18c ± 0.07
pyrroles 1-furfurylpyrrole
1829
A
0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00 2.07d ± 0.04
3.59a ± 0.09
2.47b ± 0.05
2.18c ± 0.07
2.10d ± 0.04
pyrrole
1511
A
0.00d ± 0.00 0.13c ± 0.01 0.26b ± 0.04 0.00d ± 0.00
0.39a ± 0.02
0.24b ± 0.03
0.14c ± 0.01
0.27b ± 0.01
3-methyl-1H-pyrrole
1675
B
0.00b ± 0.00 0.00b ± 0.00 0.14a ± 0.01 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
2-methylpyrazine
1265
B
0.00g ± 0.00 0.44f ± 0.02 0.57ef ± 0.07 2.38c ± 0.03
2.98a ± 0.03
1.22d ± 0.01
0.69e ± 0.01
2.79b ± 0.11
2,5-dimethylpyrazine
1319 A,B 0.07g ± 0.01 3.65f ± 0.21 4.18e ± 0.16 0.00h ± 0.00
8.71b ± 0.04
7.00c ± 0.12
6.39d ± 0.04
9.56a ± 0.10
2,6-dimethylpyrazine
1320 A,B 0.00f ± 0.00 0.80e ± 0.02 0.95d ± 0.05 12.32a ± 0.01
0.00f ± 0.00
0.98c ± 0.02
0.00f ± 0.00
1.07b ± 0.06
2,3-dimethylpyrazine
1334
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.25a ± 0.03
0.14b ± 0.01
0.00c ± 0.00
2-ethyl-5-methylpyrazine
1385
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.50b ± 0.01
1.56a ± 0.05
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
trimethylpyrazine
1402 A,B 0.00h ± 0.00 0.03g ± 0.02 0.11f ± 0.01 0.76b ± 0.05
1.10a ± 0.02
0.50c ± 0.02
0.39e ± 0.04
0.44d ± 0.02
acetylpyrazine
1629
A
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.16b ± 0.01
0.22a ± 0.02
1077
A
0.00f ± 0.00 0.32e ± 0.01 0.53d ± 0.02 0.00f ± 0.00
1.38b ± 0.01
0.72c ± 0.02
1.71a ± 0.03
0.77c ± 0.06
pyrazines
thioethers dimethyl disulfide
34
dimethyl trisulfide
1358
B
0.00h ± 0.00 0.25g ± 0.01 0.29e ± 0.01 0.63c ± 0.01
0.71b ± 0.01
0.29f ± 0.02
0.78a ± 0.02
0.41d ± 0.01
furfuryl methyl disulfide
1066
B
0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00
0.96a ± 0.01
0.26b ± 0.01
0.12d ± 0.02
0.16c ± 0.01
hexanal
1083
B
0.00f ± 0.00 0.00f ± 0.00 0.27e ± 0.01 0.00f ± 0.00
0.47d ± 0.02
0.85b ± 0.01
1.14a ± 0.01
0.62c ± 0.01
heptanal
1182
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.23b ± 0.01
0.39a ± 0.01
0.00c ± 0.00
octanal
1281
C
0.41c ± 0.01 0.00g ± 0.00 0.13f ± 0.01 0.63a ± 0.01
0.00g ± 0.00
0.27e ± 0.05
0.58b ± 0.04
0.36d ± 0.06
nonanal
1382
B
1.80c ± 0.04 1.02g ± 0.02 0.95h ± 0.02 2.45a ± 0.04
1.51d ± 0.06
1.37f ± 0.03
2.18b ± 0.01
1.41e ± 0.01
benzaldehyde
1520 A,B 3.24e ± 0.02 0.00h ± 0.00 0.41g ± 0.01 4.50b ± 0.04
3.78d ± 0.02
5.08a ± 0.03
4.37c ± 0.01
1.36f ± 0.01
AB 0.00g ± 0.00 0.35f ± 0.01 0.44ef ± 0.03 35.53a ± 0.09
15.77b ± 0.15
0.50e ± 0.01 10.05c ± 0.05
6.79d ± 0.03
aldehydes
acids acetic acid
1448
butanoic acid
1631
B
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 1.22a ± 0.10
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
benzoic acid
1644
C
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.10a ± 0.01
0.00b ± 0.00
0.00b ± 0.00
octanoic acid
2062 A,B 0.00g ± 0.00 0.95e ± 0.01 1.07e ± 0.05 2.80a ± 0.04
2.01c ± 0.04
1.42d ± 0.03
0.53f ± 0.01
2.31b ± 0.08
nonanoic acid
2171
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.22a ± 0.01
0.00b ± 0.00
0.00b ± 0.00
decanoic acid
2276
C
0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00 2.27a ± 0.09
1.22b ± 0.01
0.69c ± 0.01
0.44d ± 0.01
1.21b ± 0.04
acetic acid ethenyl ester
981
B
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.51a ± 0.01
0.00b ± 0.00
3-methylbutyl acetate
1122
B
0.00e ± 0.00 0.02d ± 0.01 0.03c ± 0.01 0.00e ± 0.00
0.00e ± 0.00
0.06b ± 0.01
0.06b ± 0.01
0.55a ± 0.04
terpinyl acetate
1693
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.06a ± 0.01
0.00b ± 0.00
0.00b ± 0.00
furfuryl acetate
1528
A
0.00f ± 0.00 0.13e ± 0.01 0.22c ± 0.01 0.37a ± 0.01
0.37a ± 0.01
0.28b ± 0.01
0.22d ± 0.02
0.22d ± 0.02
A,B 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
1.39a ± 0.01
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00g ± 0.00 1.57f ± 0.02 1.84e ± 0.04 3.11d ± 0.04
5.67b ± 0.01
3.33c ± 0.01
1.54f ± 0.01
10.79a ± 0.04
esters
alcohols 1-hexanol
1340
linalool
1525
B
35
1547
C
0.34c ± 0.01 0.63b ± 0.01 1.13a ± 0.02 0.00f ± 0.00
0.60b ± 0.01
0.23d ± 0.01
0.14e ± 0.01
0.22d ± 0.01
(2E)-3,7-dimethyl-2,6-octadien-1-ol 1844
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.16a ± 0.01
benzyl alcohol
1880
A
0.00f ± 0.00 0.07f ± 0.01 0.09d ± 0.01 0.46b ± 0.02
0.55a ± 0.01
0.10d ± 0.01
0.11d ± 0.03
0.25c ± 0.01
phenylethyl alcohol
1920
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.60a ± 0.01
0.31b ± 0.01
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
1-butanol
1154
A
0.00b ± 0.00 0.00b ± 0.00 1.26a ± 0.01 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
1-pentanol
1246
B
0.00b ± 0.00 0.00b ± 0.00 0.21a ± 0.01 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
1-heptanol
1451
A
0.20b ± 0.01 0.00c ± 0.00 1.08a ± 0.06 0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
α-terpineol
1698
B
0.00b ± 0.00 0.21a ± 0.01 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
phenol
2020
B
0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00 0.11ab ± 0.01
0.12a ± 0.01
0.06c ± 0.01 0.11ab ± 0.01 0.11b ± 0.01
2-butanone
917
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
3.80a ± 0.02
2.83b ± 0.01
0.00c ± 0.00
2-pentanone
980
B
0.00d ± 0.00 0.06c ± 0.01 0.07b ± 0.01 0.00d ± 0.00
0.00d ± 0.00
0.80a ± 0.02
0.00d ± 0.00
0.00d ± 0.00
2,3-pentanedione
1061
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.26a ± 0.02
0.15b ± 0.01
0.00c ± 0.00
3-penten-2-one
1132
B
0.00e ± 0.00 0.10d ± 0.01 0.22c ± 0.02 0.00e ± 0.00
0.00e ± 0.00
0.256b ± 0.01 0.50a ± 0.01
0.00e ± 0.00
4-methyl-3-penten-2-one
1134
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
1.33b ± 0.04
0.00c ± 0.00
0.00c ± 0.00
6.33a ± 0.12
2-heptanone
1172
B
0.00f ± 0.00 0.06e ± 0.01 0.09d ± 0.01 0.00f ± 0.00
0.13c ± 0.01
0.22b ± 0.01
0.11d ± 0.01
0.88a ± 0.02
2-octanone
1272
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.11b ± 0.01
0.11b ± 0.01
0.21a ± 0.01
2-nonanone
1378
B
0.21b ± 0.01 0.06d ± 0.01 0.07d ± 0.01 0.00e ± 0.00
0.00e ± 0.00
0.11c ± 0.01
0.00e ± 0.00
0.25a ± 0.01
2-nonen-4-one
1476
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.15a ± 0.01
1-phenylethanone
1651
A
0.00d ± 0.00 0.85a ± 0.02 0.56b ± 0.01 0.27c ± 0.01
0.00d ± 0.00
0.00d ± 0.00
0.00d ± 0.00
0.00d ± 0.00
6-methyl-5-hepten-2-one
1300
B
0.00b ± 0.00 0.07a ± 0.01 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
1-octanol
ketones
alkenes
36
β-myrcene
1141
B
0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00
0.55b ± 0.01
0.37c ± 0.01
0.19d ± 0.01
2.68a ± 0.21
δ-3-carene
1145
A
0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00
0.18c ± 0.01
1.42a ± 0.02
0.90b ± 0.01
0.00d ± 0.00
dl-limonene
1166
B
0.00f ± 0.00 0.34e ± 0.01 0.53d ± 0.01 0.54d ± 0.01
0.36e ± 0.01
1.81b ± 0.02
1.60c ± 0.01
2.23a ± 0.01
trans-β-ocimene
1213
A
0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00
0.66b ± 0.01
0.11c ± 0.01
0.00d ± 0.00
0.81a ± 0.01
γ-terpinene
1230
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.25a ± 0.01
D-limonene
1196
B
1.12a ± 0.03 0.34b ± 0.01 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
p-cymene
1256
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00 0.00b ± 0.00
0.45a ± 0.01
limonene
1188
B
0.00b ± 0.00 0.00b ± 0.00 1.66a ± 0.01 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
styrene
1237
B
0.00c ± 0.00 0.21b ± 0.01 1.03a ± 0.01 0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
x
Kovats indices of compounds on HP - INNOWAX column.
y
Identification method of the compounds: (A) mass spectrum and Kovats index according to literature (Niu et al., 2011; Xiao et al., 2011; Xiao et al.,
2014); (B) mass spectrum compared with databases； （C）mass spectrum and Kovats index agree with that of the authentic compound run under the same conditions of the samples. z
Results were expressed as mean value ± standard deviation (n = 2).
Values bearing different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
37
Highlights A procedure was set up to produce extract from beef bone by lipase pretreatment. An excellent beef aroma was formed due to plentiful amino acids in the extract. Industrial beef by-product was used to produce food flavouring by Maillard reaction.
38
S0308-8146(16)30434-4 http://dx.doi.org/10.1016/j.foodchem.2016.03.062 FOCH 18946
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
24 January 2016 13 March 2016 18 March 2016
Please cite this article as: Song, S., Li, S., Fan, L., Hayat, K., Xiao, Z., Chen, L., Tang, Q., A novel method for beef bone protein extraction by lipase-pretreatment and its application in the Maillard reaction, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.03.062
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel method for beef bone protein extraction by lipase-pretreatment and its application in the Maillard reaction Running title: Lipase-pretreatment extraction of protein from beef bone Shiqing Songa, Sisi Lia, Li Fana, Khizar Hayatb, Zuobing Xiao *,a, Lihua Chen a, Qi Tanga a
School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai,
201418, PR China b
Department of Food Science and Nutrition, College of Food and Agricultural Sciences,
King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia *
Corresponding author: +86-21-60873150; Fax: +86-21-60873424
E-mail: [email protected]
1
ABSTRACT: Five beef bone hydrolysates were obtained by different enzyme treatment schemes, including papain (M), combination of porcine pancreatic lipase and papain (Z+M, combination of lipase and papain (Y+M), Protamex (F), combination of porcine pancreatic lipase and Protamex(Z+F). The degree of hydrolysis (DH), free amino acids and molecular weight distribution of these hydrolysates were evaluated. To further explore the differences between these five hydrolysates, Maillard reaction
products
(MRPs)
were
prepared
using
a
xylose/cysteine/hydrolysate model. It was found that the DH, content of low molecular weight peptides and amino acids of hydrolysates increased significantly after lipase pre-treatment. GC-MS showed that the total content of furans, pyrroles and thioethers in MRPs Y+M increased by 78.0% compared with MRPs M, while in MRPs Z+F, pyrazines increased by 44.1% compared with MRPs F. Examining the sensory characteristics of the MRPs, the MRP from the hydrolysate of Y+M had the best mouthful, umami and meaty characteristics. The correlation analysis further confirmed that an appropriate lipase pre-treatment could improve the flavour of MRPs. Keywords:
Bone
hydrolysates;
lipase;
Maillard
reaction
product;
Descriptive sensory analysis; GC-MS; partial least squares regression. Chemical compounds studied in this article: 2
Xylose (PubChem CID: 135191); Cysteine (PubChem CID: 5862); Dimethyl disulfide (PubChem CID: 12232); Dimethyl trisulfide (PubChem CID: 19310); 2-Pentylfuran (PubChem CID: 19602); Hexanal (PubChem CID: 6184)
3
1. Introduction Approximately 48.28 million cattle were slaughtered in China in 2013 (State Statistical Bureau, 2014). Most of this beef is deboned in plant and the meat is sold as packaged beef. During this process, as much as 6‒12% (based on carcase weight) of bone was left. This could result in approximately 4.43 million tonnes of bone (almost no meat attached to the bone) disposed of or sold at a lower price as inedible by-products (Wang et al., 2015). Beef bone contains a notable amount of muscle, connective tissue and fat, and therefore represents a valuable source of proteins, containing about 47% moisture, 21% protein (collagen), 15% fat, and 15% ash. Non-utilisation or underutilisation of animal by-products not only leads to loss of potential revenues but also leads to a higher cost of disposal of these products. For that reason, industries have begun to develop various technologies to make use of this waste, mainly in the form of value-added products, at the same time reducing the cost derived from its disposal. Continuous efforts have been made to improve the functional and nutritional value of bone. Boles et al. (2000) used different solutions (4% sodium chloride, 4% sodium chloride with either 0.3 M sodium tripolyphosphate, tetrasodium pyrophosphate or 0.05 M NaOH to effectively extract proteins from beef bones. These proteins could be used to manufacture finely comminuted sausage products with similar texture to 4
sausages made with commercially available proteins. Nikolaev et al. (2008) stated that a functional meat protein could be obtained by the fermentation of meat-bone broiler residues. Recently, enzymatic hydrolysis was employed to extract proteins and produce peptides (Morimura et al., 2002), which formed an effective way to recover proteins from the by-products of animal processing. Unlike acidic or alkaline hydrolysis, enzymatic proteolysis is mild and controllable, which helps to improve the quality and functional properties of protein (Kristinsson & Rasco, 2000). Linder et al. (1995) reported that the utilisation of bone mainly focused on the enzymatic extraction of nutrients. After hydrolysis, bone could be developed into value-added products. Linder et al. (1997) also described that the enzymatic hydrolysate of veal bone contained a large amount of glycine and proline, whose nutritional value was much higher than the hydrolysate treated by acid or alkali. It was found that the hydrolysates were useful for soups, sauces and gravies. Dong et al. (2014) used hot-pressure combined with enzymolysis to extract protein from chicken bone; these hydrolysates demonstrated a new kind of potential suitable nutritional supplement in various foods. Other researchers also found that the hydrolysed protein was an important flavouring agent (Lieske & Konrad, 1994; Lafarga & Hayes, 2014; Zhan et al., 2013), which could give Maillard reaction products (MRPs) with lifelike meat flavour (Pommer, 1995). 5
The water-soluble meat flavour precursors consist of free amino acids, peptides, and reducing sugars (Khan, Jo, & Tariq, 2015). Madruga et al. (2010) reported that by controlling the degree of hydrolysis (DH), different constituents of these precursors could generate different flavours. In general, the lower the DH is, the fewer the precursors. It is well-known that beef bone is usually surrounded by adipose tissue, which may prevent the combination of protein and protease, leading to a low degree of hydrolysis. Linder et al. (1997) hydrolysed veal bone using Neutrase only, with an unsatisfactory DH. Therefore, it is necessary to develop an effective way for better utilisation of beef bone by-product. Since lipase could hydrolyse redundant adipose tissue during lean meat processing, this enzyme was chosen to pre-treat the beef bone. The objective of present study is to develop a new method for the preparation of protein from beef bone, and compare it with other methods. The study includes(A) analysis of the degree of hydrolysis, free amino acids and molecular weight of five bone hydrolysates hydrolysed by different treatment schemes, including papain, combination of porcine pancreatic lipase and papain, combination of lipase and papain, Protamex, combination of porcine pancreatic lipase and Protamex;
(B) Comparison of the sensory
characteristics and the volatile compounds of the MRPs prepared from the five hydrolysates. (C) Study of the relationship between free amino acids, 6
molecular weight distribution of hydrolysates and the sensory characteristics of MRPs. Through the above analyses, the influence of the lipase pre-treatment on beef bone hydrolysate and MRP was investigated. 2. Materials and methods 2.1. Chemicals and materials Beef bone was purchased from Shanghai Tesco Supermarket (Shanghai, China). Xylose and cysteine were purchased from Sigma China Co., Ltd. (Beijing, China). Papain (2000 U/mg) and Protamex (31.4 U/mg) were obtained from Novo Co., Ltd. (Novozyme Nordisk, Bagsvaerd, Denmark). Porcine pancreatic lipase (20 U/mg) was purchased from Shanghai Source Poly Biological Technology Co., Ltd. (Shanghai, China). Lipase (Yiming, 20 U/mg) was purchased from Yiming Biological Products Co., Ltd (Jiangsu, China). Formaldehyde and NaOH were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The other chemical reagents were purchased from National Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Preparation of bone hydrolysates hydrolysed by different enzymes Beef bones were first cleaned of meat, fat and bone marrow and heated for 4 h at 121 °C, 0.1MPa a in pressure vapour steriliser (Shanghai Shenan Medical Devices Co., Ltd., Shanghai, China). Then it was dried at 60 °C for 4 h before grinding into powder (80 mesh size) by high-speed grinding machine (Tianjin Instrument Co., Ltd., Tianjin, China). Bone powder was 7
mixed with deionised water in a certain proportion. Then the mixture was hydrolysed by different enzymes. The preparations of five different beef bone hydrolysates are listed in Table 1. All five hydrolysates were prepared at the optimal conditions of the enzymes. The composite enzymatic hydrolysates were hydrolysed by porcine pancreatic lipase or lipase for 3 h, followed by papain or Protamex for 3 h. After the enzyme deactivation at 90 ℃ for 10 min, these five hydrolysates (designated M, F, Z+M, Z+F, Y+M,) were centrifuged (Scientific Instrument Co., Ltd., Shanghai, China) at 4000 g for 20 min. The supernatants were kept at 4 ℃ until used. 2.3. Determination of DH DH of the hydrolysates was measured according to Song et al. (2013). 2.4. Free amino acid analysis A pre-treatment of the sample was needed before the amino acid analysis. For the determination of free amino acids, 5 mL sample were added to a volumetric flask (25 mL), and 5% trichloroacetic acid (TCA) was added to volume, to precipitate peptides or proteins (Song et al., 2013). The solution was filtered through Whatman filter paper No.4 after incubation for 2 h at room temperature. Then the filtrate was centrifuged (Scientific Instrument Co., Ltd., Shanghai, China) at 12300 g for 10 min and stored at 4 ℃. Amino acids in bone hydrolysates were analysed (Song et al., 2013; Liu et al., 2012). The sample (20 µL) was injected into an automated online 8
derivatisation system with and analyzed by an Agilent 1100 HPLC with UV detector operated at 338 nm/262 nm using a Hypersil ODS column（4.6 mm × 250 mm × 5 µm）, while the flow rate was 1 mL/ min. Temperature of the column was 40 ℃. The mobile phase was composed of 0.6 mM sodium acetate and 0.15 mM sodium acetate: acetonitrile: methanol, (1:2:2, v:v:v). Amino acidsstandards (Sigma Chemical Co., St. Louis, MO) were used for the determination of a calibration curve for calculation, and the identification and quantification were decided on the basis of the retention time and the peak area of standard compounds, respectively. 2.5. Molecular weight distribution analysis The molecular weight distribution of the bone hydrolysates was analysed by a Waters 600 liquid chromatography system (Waters Co., Milford, MA,). The system was equipped with a Waters 2487 UV detector and Empower work station on a 2000 (300 × 7.8 mm) SWXL TSK gel filtration column (Tosoh
Co.,
Tokyo,
Japan).
The
mobile
phase,
consisting
of
acetonitrile/water/trifluoroacetic acid (45:55:0.1, v:v:v), was delivered at a flow rate of 0.5 mL/ min. The column temperature was 30 ℃ and 10 µL sample were injected into the HPLC system. The molecular weight calibration curve was obtained using five standards from Sigma: cytochrome C (12500 Da), aprotinin (6500 Da), bacitracin (1450 Da), tetrapeptide GGYR (451 Da), and tripeptide GGG (189 Da). The chromatogram was 9
recorded using a UV detector at 220 nm. The data were analysed by gel permeation chromatography (GPC) software. 2.6. Preparation of MRPs The supernatants of hydrolysates were concentrated by a rotary evaporator till the solids content was 20%. Then cysteine (0.4 g) and xylose (0.64 g) were added into 20 mL concentrated supernatant. The mixture was adjusted to pH 7.0 with 6 M NaOH, then transferred into a reaction vessel and allowed to react in an oil bath with magnetic stirring (130 rpm) at 110 °C for 90 min. These five Maillard reaction products (marked as MRPs M, F, Z+M, Z+F, Y+M, respectively) were immediately cooled in ice-water and stored in a refrigerator (‒18 °C) for further analyses. Three control samples were prepared from (1) hydrolysate of beef bone, (2) cysteine and xylose, beef bone powder, and (3) cysteine and xylose, respectively. Their preparation conditions were treated as mentioned above, and they were named M, MRPs 1 and MRPs 2, respectively. 2.7. Sensory characteristics of MRPs To ascertain the sensory characteristics of these five Maillard reaction products, sensory evaluation was carried out according to the method of Schlichtherle-Cerny
and
Amadò
with
some
modifications
(Schlichtherle-Cerny & Amadò, 2002). The evaluation of the samples was done by a panel of five males and three females (23‒45 years old) from the 10
School of Perfume and Aroma Technology at Shanghai Institute of Technology, China. All panellists had numerous experiences in sensory evaluation of foods. The eight panellists had previously been trained for 3 h to define descriptive terms and to decide suitable reference solutions for the samples. The reference solutions were prepared as follows: 2.0 g Hershey’s syrup caramel (Hershey (Shanghai) Co., Ltd) in 40 mL water was labelled caramel-like, 3 mM MSG (Shanghai Totole Food Co., Ltd) was labelled umami, 15 mM lactic acid (Henan Jindan Technology Co., Ltd) was labelled sour, defatted brisket meat (0.5 kg, 2.5-cm thick, purchased from Wal-Mart supermarket) boiled in water (1 kg) for 2 h was labelled meat aroma; 2.0 mM caffeine (Illycaffe (Shanghai) Trading Co., Ltd) was labelled bitter, and 10 g bouillon cube (beef flavour consisting of MSG, yeast extract, and beef extract), dissolved in water was labelled mouthful. The evaluation was implemented using a 1‒10 interval scale (0 = none, 10 = extremely strong), where 5 points was the score of the reference solutions. The sensory evaluation took place in a sensory laboratory equipped to international standards. Maillard reaction products (60 mL) and 60 mL reference solutions were tasted at 22 ± 2 ℃ in separated sensory booths at the same time. 2.8. GC-MS analysis The volatile compounds were extracted by SPME with a 75 µm Carboxen-PDMS fibre (Supelco, Bellefonte, PA). Maillard reaction product 11
(10.0 g) was placed into a glass vial and 50 µL internal standard 1,2-dichlorobenzene in methanol (0.5 µg/µL) was added into the sample before trapping. The glass vial was sealed with a lid and placed in a water bath at 55 ℃ for 30 min with aging the SPME fibre in the headspace to allow the equilibration of the volatile compounds. Then the fibre was desorbed at 250 ℃ for 5 min. Separation of the volatile compounds was implemented on an HP-INNOWAX (60 m × 0.25 mm, 0.25 µm; J & W Scientific, Folsom, CA) capillary column. Helium was used as the carrier gas, flowing at 1.8 mL/min. The starting temperature of the chromatographic column was 50 ℃, held for 3 min, then increased up to 230 ℃ at 3 ℃ / min, and then held at this temperature for 7 min. The mass spectrometric detector was operated at an electron voltage of 70 eV and the temperature of the ion source was 230 ℃. Volatile compounds were identified by comparing the detector data (Kovats retention Indices, KI) of samples with authentic standards, the published literature, the NIST 11 and the Wiley 07 databases. The KI values were calculated relative to an n-alkanes series (C8‒C23) under the same condition of the samples. Approximate quantities of the volatile compounds were estimated by comparison of their peak areas with that of the internal standard, obtained from the total ion chromatograms, assuming that the relative response factor was 1 and the recovery ratio was 100%. The quantitative formula was as follows: 12
Wi = f ' *
Ai * ms /m As
where Ai is the peak area of compound i, As is the peak area of internal standard, ms is the mass of internal standard, m is the mass of sample, f’ is a relative correction factor, assumed to be 1, Wi is the concentration (µg/g) of compound i. 2.9. Statistical analysis Data from DH, free amino acids and sensory evaluation were subjected to analysis of variance by SPSS version 13.0 (SPSS Inc., Chicago, IL). Partial least squares regression (PLSR) was used to obtain the potential correlations among free amino acids, molecular weight distribution and sensory characteristics. All variables were centred and scaled to 1/SD so that each variable has a unit variance and zero mean before applying PLSR analysis, and to obtain an unbiased contribution of each variable to the criterion. All regression models were validated by full cross-validation. Multivariate analysis was performed with Unscrambler version 9.7 (CAMO ASA, Oslo, Norway). 3. Results and discussion 3.1. Effects of lipase pretreatment on DH of bone hydrolysates The protein hydrolysates of beef bone were prepared according to the method as mentioned above. The beef bone was hydrolysed by papain,
13
combination of porcine pancreatic lipase and papain, and combination of lipase and papain. The corresponding DH was 15.43 %, 23.17 % and 19.17 %, respectively (Table 1). DH values of these three bone hydrolysates were significantly different. It was found that DH of hydrolysates Z+M and Y+M were significantly higher than that of the hydrolysate which was treated with papain alone. Meanwhile, the porcine pancreatic lipase was more efficient in increasing the DH of the hydrolysate than lipase. In addition, another experiment using Protamex alone and combination of porcine pancreatic lipase and Protamex was carried out. The result indicated that DH increased greatly after the porcine pancreatic lipase pre-treatment of the samples (DH values of F and Z+F were 12.71% and 21.28%, respectively). From the above results, it is clear that the lipase pre-treatment exerted a considerable influence on the increase of DH. The possible reason was that after the lipase pre-treatment, more cleavage points were exposed, which made the interaction between proteins and enzymes easier; as a result the DH value increased. Moreover, compared with Z+F, the DH of Z+M was somewhat higher. 3.2. Analysis of free amino acids It is known that the composition and the content of the free amino acids have an important influence on the flavour properties of Maillard reaction 14
products directly and indirectly (Lorenzen, Davuluri, Adhikari, & Grün, 2005). As shown in Table 2, the lipase pre-treatment had an important influence on the content of free amino acids. The total free amino acids content of the samples hydrolysed by combination of lipase and protease significantly increased to that hydrolysed by protease alone. The total free amino acids of M, Z+M, Y+M, F and Z+F were 152 mg/g, 534 mg/g, 505 mg/g, 105 mg/g and 506 mg/g, respectively. It was obvious that Arg, Gly, Ala, Lys and Tyr were present at high proportion in all samples (Table 2). Griffith and Hammond (1989) reported that Lys could form alkylpyrazines and 2-acetyl-1-pyrroline through thermal reactions, while some potent flavour compounds are formed from cysteine and ribose through the Maillard reaction (Chen et al., 2000; Cerny, & Davidek, 2004; Yu et al., 2012). Meanwhile, enzymatic bone hydrolysates contain a larger portion of peptides than that of free amino acids, owing to the higher selectivity and specificity of enzymes. Some peptides could affect the flavour and aroma of the hydrolysate and the Maillard reaction products. It was reported that peptides of different structures and lengths could provide unique taste properties, such as umami, sour, bitter, salty and sweet (Spurvey et al., 1998). Val, Leu, Ile, Met, Phe, Ser, Arg and His are bitter amino acids; among these 8 bitter amino acids, Arg, His and Met are strongest in bitterness (Yang & Liu, 1983; Lan et al., 2010). Sample Z+F had a high content of all these 15
bitter amino acids. Umami taste was provided by Glu and hydrophilic amino acid residues (Arai et al., 1972; Arai et al., 1973). After the lipase pretreatment, the Glu decreased in Z+M, Y+M and Z+F, compared with M and F. Sour taste was closely related to umami. Kirimura et al. (1969) reported that Gly-Asp, Ala-Glu, Glu- Leu and other peptides had sour taste. 3.3. Analysis of molecular weight distribution The molecular weight distributions of five bone hydrolysates are shown in Table 3, which displays that these samples were composed of a series of low molecular weight polypeptides, especially peptides with molecular weight less than 1000 Da. As the DH increased, amino acids and some peptides less than 180 Da gradually increased. By contrast, the tendency of peptides above 500 Da was converse to that of those less than 180 Da. However, no apparent tendency was found in the peptides from 180 Da to 500 Da. Ogasawara et al. (2006a, 2006b) reported that the main compounds that provided special flavour were Maillard peptides whose molecular weight ranged from 1000 Da to 5000 Da. It was found that Maillard peptides decreased after the lipase pre-treatment. Nevertheless, by the cross linking of peptides below 1000 Da during the Maillard reaction, the content of high molecular weight peptides increased (Zamora & Hidalgo, 2005), particularly the Maillard peptides (Lan al., 2010). Because these peptides contribute to the mouthful flavour, the increase of these peptides could improve the 16
mouthful flavour and improve the thin taste of the product. 3.4. Sensory analysis of the MRPs The five Maillard reaction products were described as caramel-like, umami, sour, meaty, bitter and mouthful. An analysis of variance for MRPs was made, three replicates were applied to sensory data to assess the results. Significant differences among samples (p ≤ 0.05) for all attributes indicated that the samples had different aroma intensities. The mean intensity values of the 6 attributes and the results of Duncan’s multiple comparison tests are shown in Table 4. Comparing these five MRPs, MRPs F, Z+M, Z+F had a stronger caramel-like flavour, possibly because they were rich in furans (Van Boekel, 2006). Owing to the decrease of the bitter amino acids and the cross-linking of the peptides below 1000 Da (Lan et al., 2010), MRPs M, and F were more bitter than MRPs Z+M, Y+M and Z+F. Sour attribute increased after the lipase pre-treatment. MRPs
Y+M presented the highest
meaty, umami and mouthful attributes. Ogasawara et al. (2006b) reported that the cross-linking of peptides below 1000 Da could enhance mouthful and umami tastes. However, it was noted that MRPs Z+M and Z+F were much weaker in mouthful and umami than MRPs M and F. 3.5. GC-MS analysis of MRPs The volatile compounds of these five Maillard reaction products and three test samples (M, MRPs 1 and MRPs 2) were detected by GC-MS and 17
the analysis results are shown in Table 5. The lipase pre-treatment affected both the number of volatile compounds and the aroma intensity. A total of 64 compounds were detected and identified, including furans, pyrazines, thioethers, aldehydes and others that might have no contribution to the aroma characteristics of these eight samples. As shown in Table 5, few volatiles were detected in M, MRPs 1 and MRPs 2. M had the fewer kinds and amount of volatiles, than MRPs 1 and MRPs 2. More volatiles were detected in MRPs 2 than in MRPs 1. Furans and pyrazines were the dominant volatile compounds in the other five MRPs. After the lipase pretreatment, it was observed that the total content of furans, pyrroles and thioethers in MRPs Z+M and Y+M increased by 30.1% and 78.0%, pyrazines decreased by 10.5% and 38.7%, respectively, compared with MRPs M. In MRPs Z+F pyrazines increased by 44.1%, furans and thioethers decreased by 45.5% and 51.8%, respectively, compared with MRPs F. It was known that meaty flavour was associated with nitrogenous, sulphur-containing heterocyclic compounds and their derivatives (Song et al. 2013). Van Boekel (2006) reported that meaty flavour was mainly from sulphur compounds. Alkylpyrazines have also been detected in meat products (Mottram, 1998). Although sulfur volatiles have been demonstrated to be responsible for meat aroma, only dimethyl disulfide, dimethyl trisulfide and furfuryl methyl disulfide were detected in these samples. 18
MRPs F and MRPs Z+M had the most thioethers; however, the flavour of these two samples were not the best. These essential meaty flavour compounds were most abundant in MRPs F. However, Table 4 showed that MRPs Y+M had the highest mouthful, umami and meaty characteristics, while the overall flavour of MRPs F was not favoured. One reason might be that these aroma compounds were not contributing to the beef flavour. Another possible reason could result from the high content of furans, which possessed high caramel-like flavour, decreasing the meaty aroma. 3.6. Relationship between free amino acids, molecular weight of hydrolysates and sensory characteristics of MRPs To investigate which chemical parameters have a great effect on the flavour of the MRPs, ANOVA with PLSR was used to study the main scores accumulated from sensory evaluation and chemical parameters (free amino acids and molecular weights). The X matrix was designated to free amino acids and molecular weight; the Y matrix was designated to sensory attributes and the five MRPs (Fig. 1). The optimal number of components in the ANOVA- PLSR model presented was determined to be 3 Principal components PC1 versus PC2, PC1 versus PC3, and PC2 versus PC3 were explored, but PC1 versus PC3 is not presented here, as additional information was not obtained through this plot. Further PCs did not provide any predictive improvement on the Y matrix obtained. In this model session, 19
level effects were removed and the data were normalised through mean centring. The calibrated explained variances for these models were PC 1 = 59 %, PC 2 = 28 % and PC 3 = 10 %, respectively. As indicated from Fig. 1A, MRPs M and MRPs F were located on the left side, MRPs F correlated with peptides above 500 Da, and four amino acids Ala, Asp, Glu and Gly. MRPs M correlated with Met, Val, His, Phe, Leu, Ile and Arg. These amino acids are associated with bitter attributes to a great extent. Our results are consistent with the finding of Lan et al.(2010). MRPs Z+M, MRPs Y+M and MRPs Z+F were located on the right side and were significantly distinguishable from MRPs F and MRPs M. Thedifferent enzymatic treatments of beef bone had a significant influence on Maillard reaction products flavour. Further information can be obtained from Fig. 1B. Sample MRPs F is on the left side, which also correlated with amino acids Ser and Cys-S, sour and caramel-like aromas. MRPs Y+M and MRPs Z+M were located on the right side and they were associated with Lys and Arg. as well as with peptides of molecular weight less than 180 Da, umami, meaty, bitter and mouthful attributes. The sensory evaluation results also showed (Table 3) that MRPs Y+M had the highest score in umami, meaty and mouthful attributes. MRPs Z+F correlated with His, Phe, Val, Met, Leu, Ile and peptides from 180 Da to 500 Da. From the above results, it was observed that the aroma and flavour 20
of the MRPs could be improved by an appropriate pre-treatment of lipase. 4. Conclusions This study clearly revealed that lipase pre-treatment was efficient in increasing DH, the content of peptides below 1000 Da and free amino acids of bone hydrolysates. After the cross-linking of sugars and amino acids through the Maillard reaction, the aroma and flavour of Maillard reaction product from added bone hydrolysate by the pre-treatment of a combination of lipase and papain had the best taste with mouthful, umami and meaty characteristics. Further investigation by GC- MS showed that the volatile compounds increased, when compared with Maillard reaction products from bone hydrolysate prepared using papain alone. Meanwhile, PLSR analysis among molecular weight, free amino acids and sensory attributes of MRPs clearly showed that an appropriate pre-treatment of lipase could improve the flavour through thermal reactions. Hence, the protein hydrolysate of lipase and papain could be used to generate beef flavour. Acknowledgements The research was supported in part by National Natural Science Foundation of China (31201415), School project of Shanghai Institute of Technology (YJ2013-22) and Shanghai Engineering Technology Research Center of Fragrance and Flavour (12DZ2251400). References 21
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aromatic fingerprinting using HS-SPME-GC-MS and multivariate analysis. Journal of Food Science, 79, 284-294. Yang, B. G., & Liu, S. C. (1983). Studies on the bitterness of protein. Food Science, 12, 1-2. Yu, A. N., Tan, Z. W., & Wang, F. S. (2012). Mechanism of formation of sulphur aroma compounds from L-ascorbic acid and L-cysteine during the Maillard reaction. Food chemistry, 132,1316-1323. Zamora, R., & Hidalgo, F. J. (2005). Coordinate contribution of lipid oxidation and Maillard reaction to the nonenzymatic food browning. Critical Reviews in Food Science and Nutrition, 45,49-59. Zhan, P., Tian, H., Zhang, X., & Wang, L. (2013). Contribution to aroma characteristics of mutton process flavor from the enzymatic hydrolysate of sheep bone protein assessed by descriptive sensory analysis and gas chromatography olfactometry. Journal of Chromatography B, 921,1-8. Figure Captions Fig.1. The overview of the variation found in the mean data from the PLSR correlation loadings plot for five MRPs. The loadings of the X- and Y-variables for (A) PC 1 versus PC 2, (B) PC 2 versus PC 3 are presented. The model was derived from free amino acids and molecular weight of peptides of bone hydrolysates as the X matrix and sensory attributes of MRPs as Y matrix. Elipses represent r2 = 0.5 and 1.0. 27
Fig. 1
29
Table 1 Preparation of five different beef bone hydrolysates hydrolysates
M
Z＋M
Y＋M
optimum conditions of lipase pretreatment
—
porcine pancreatic lipase temperature: 35 ℃; pH = 7.0; time: 3 h; enzyme/substrate ratio 1.5% (w/w) lipase temperature: 35 ℃; pH = 7.5; time: 3 h; enzyme/substrate ratio 1.5% (w/w)
F
—
Z＋F
porcine pancreatic lipase temperature: 35 ℃; pH = 7.0; time: 3 h; enzyme/substrate ratio 1.5% (w/w)
optimum conditions of protein treatment papain temperature: 60 ℃; pH 3 h; enzyme/substrate (w/w) papain temperature: 60 ℃; pH 3 h; enzyme/substrate (w/w) papain temperature: 60 ℃; pH 3 h; enzyme/substrate (w/w)
degree of hydrolysis (%)
= 6.0; time: ratio: 1.0%
15.43b ± 0.05
= 6.0; time: ratio: 1.0%
23.17e ± 0.11
= 6.0; time: ratio: 1.0%
19.17c ± 0.12
Protamex Temperature: 40 ℃; pH= 6.5; Time: 3 h; Enzyme/substrate ratio: 1.0% (w/w) Protamex Temperature: 40 ℃; pH= 6.5; Time: 3 h; Enzyme/substrate ratio: 1.0% (w/w)
12.71a ± 0.01
21.28d ± 0.16
Values bearing different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
30
Table 2 Free amino acid analysis of the five bone hydrolysates (mean ± SDx ) concentration (mg/g) amino acid
M a
Asp Glu Ser His Gly Thr Arg Ala Tyr Cys-S Val Met Phe Ile Leu Lys Pro x
8.56 0.01 14.68e 0.03 1.71a 0.01 3.83b 0.02 31.91b 0.01 2.53a 0.01 34.76b 0.04 13.45b 0.02 1.53a 0.01 1.69a 0.01 6.10b 0.01 5.42b 0.01 8.68b 0.02 6.38b 0.01 5.78b 0.05 4.59b 0.03 0.35b 0.01
Z+M d
± 19.51 0.01 ± 6.08b 0.02 ± 12.57e 0.14 ± 7.18c 0.08 ± 88.26d 1.13 ± 22.52d 0.01 ± 83.47d 1.47 ± 40.40d 0.56 ± 63.37e 0.32 ± 19.34d 0.19 ± 14.06d 0.01 ± 13.64c 0.01 ± 21.87c 0.45 ± 14.30c 0.11 ± 13.09c 0.01 ± 83.78e 1.19 ± 10.23e 0.01
Y+M c
± 17.42 0.01 ± 5.51a 0.01 ± 11.95d 0.03 ± 9.08d 0.02 ± 76.02c 1.16 ± 22.92d 0.71 ± 82.56c 1.55 ± 36.17c 0.71 ± 55.03d 0.04 ± 17.03c 0.02 ± 13.79c 0.01 ± 16.17d 0.17 ± 26.65d 0.25 ± 16.58d 0.01 ± 14.70d 0.02 ± 77.48d 0.51 ± 5.63d 0.05
F b
± 9.11 0.01 ± 10.78d 0.02 ± 2.28b 0.01 ± 1.60a 0.01 ± 23.31a 0.17 ± 3.12b 0.01 ± 16.68a 0.24 ± 9.93a 0.01 ± 6.32b 0.01 ± 4.20b 0.01 ± 2.80a 0.02 ± 3.27a 0.01 ± 4.62a 0.01 ± 3.27a 0.11 ± 3.01a 0.01 ± 0.57a 0.01 ± 0.17a 0.01
Z+F
± 19.67d 0.98 ± 6.68c 0.02 ± 11.18c 0.17 ± 12.31e 0.05 ± 88.49d 1.22 ± 18.64c 0.05 ± 85.04e 0.44 ± 42.10e 0.17 ± 48.81c 0.15 ± 25.33e 0.02 ± 18.80e 0.01 ± 16.55e 0.01 ± 28.03e 0.02 ± 17.69e 0.02 ± 17.16e 0.17 ± 46.14c 0.49 ± 3.29c 0.24
Results were expressed as mean value ± standard deviation (n = 3).
Values bearing different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
31
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Table 3 Changes of molecular weight distribution (percent of total area) in five bone hydrolysates Samplesx MW(Da) M 0.03b ± >5000
Y＋M
F
Z＋F
0.02b ±
0.13a ±
0.04b ±
0.01
0.01
0.01
Z＋M c
0.00 ± 0.00 0.01 0.21c ±
0.12c ±
0.17c ±
0.71a ±
0.50b ±
0.06
0.01
0.03
0.03
0.01
1.18c ±
0.71d ±
0.83d ±
1.76a ±
1.38b ±
0.09
0.06
0.05
0.02
0.01
8.11b ±
4.46e ±
5.00d ±
10.10a ±
6.61c ±
0.08
0.01
0.05
0.02
0.02
25.85b ±
16.58d ±
16.86c ±
28.54a ±
16.38e ±
0.09
0.02
0.04
0.01
0.01
52.75c ±
58.58b ±
58.90a ±
49.27d ±
58.98a ±
0.07
0.03
0.01
0.01
0.05
12.61d ±
19.57a ±
18.28b ±
9.55e ±
16.12c ±
0.35
0.01
0.01
0.06
0.06
3000‒5000
2000‒3000
1000‒2000
500‒1000
180‒500
<180 x
Peptides in bone hydrolysates as percent of total area (%).
Values bearing different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
32
Table 4 The mean scores of the 5 attributes for the 5 MRPs in descriptive sensory evaluation sample
mean score caramel-like
umami
sour
meaty
bitter
mouthful
MRPs – Mx
5.67b
6.19d
3.78a
6.21d
7.96e
6.34d
MRPs - Z+M
7.32d
4.22a
7.92d
3.87a
6.65d
4.94b
MRPs - Y+M
4.80a
7.75e
6.88b
8.14e
4.40a
8.28e
MRPs - F
6.67c
6.08c
7.63c
5.86c
6.24c
5.64c
MRPs - Z＋F
8.12e
4.87b
8.26e
4.32b
5.32b
4.32a
x
MRPs - M/MRPs - Z+M/MRPs - Y+M/MRPs - F/MRPs - Z+F represented the Maillard reaction
products from M/Z+M/Y+M/F/Z+F, respectively. Mean scores for each attribute with different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
33
Table 5 Volatile compounds of eight MRPs analysed by GC-MS Compounds
KIx
IDy
Concentration (µg/100 g)*10 ± SDz M
MRPs - 1
MRPs - 2
MRPs - M
MRPs - Z+M
MRPs - Y+M
MRPs - F
MRPs - Z+F
furans 2-methylfuran
883
B
0.00g ± 0.00 5.48f ± 0.06 7.74e ± 0.06 7.97e ± 0.23
27.23d ± 0.09
33.99b ± 0.13 41.65a ± 0.61 33.66c ± 0.43
2-pentylfuran
1224
B
0.00f ± 0.00 0.00f ± 0.00 0.95c ± 0.02 0.53e ± 0.01
1.19b ± 0.02
0.71d ± 0.05
2.19a ± 0.16
0.00f ± 0.00
2-acetylfuran
1502
B
0.00g ± 0.00 1.07f ± 0.06 1.31e ± 0.01 1.58d ± 0.11
2.02c ± 0.03
2.32b ± 0.01
2.78a ± 0.11
2.71a ± 0.03
2-(methoxymethyl)furan
1232
A
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.17a ± 0.01
0.14b ± 0.01
0.00c ± 0.00
furfural
1443
A,B 0.00h ± 0.00 5.55f ± 0.14 1.44g ± 0.01 40.15d ± 0.17
31.64e ± 0.10
53.64b ± 0.06 97.58a ± 0.03 42.18c ± 0.07
pyrroles 1-furfurylpyrrole
1829
A
0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00 2.07d ± 0.04
3.59a ± 0.09
2.47b ± 0.05
2.18c ± 0.07
2.10d ± 0.04
pyrrole
1511
A
0.00d ± 0.00 0.13c ± 0.01 0.26b ± 0.04 0.00d ± 0.00
0.39a ± 0.02
0.24b ± 0.03
0.14c ± 0.01
0.27b ± 0.01
3-methyl-1H-pyrrole
1675
B
0.00b ± 0.00 0.00b ± 0.00 0.14a ± 0.01 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
2-methylpyrazine
1265
B
0.00g ± 0.00 0.44f ± 0.02 0.57ef ± 0.07 2.38c ± 0.03
2.98a ± 0.03
1.22d ± 0.01
0.69e ± 0.01
2.79b ± 0.11
2,5-dimethylpyrazine
1319 A,B 0.07g ± 0.01 3.65f ± 0.21 4.18e ± 0.16 0.00h ± 0.00
8.71b ± 0.04
7.00c ± 0.12
6.39d ± 0.04
9.56a ± 0.10
2,6-dimethylpyrazine
1320 A,B 0.00f ± 0.00 0.80e ± 0.02 0.95d ± 0.05 12.32a ± 0.01
0.00f ± 0.00
0.98c ± 0.02
0.00f ± 0.00
1.07b ± 0.06
2,3-dimethylpyrazine
1334
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.25a ± 0.03
0.14b ± 0.01
0.00c ± 0.00
2-ethyl-5-methylpyrazine
1385
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.50b ± 0.01
1.56a ± 0.05
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
trimethylpyrazine
1402 A,B 0.00h ± 0.00 0.03g ± 0.02 0.11f ± 0.01 0.76b ± 0.05
1.10a ± 0.02
0.50c ± 0.02
0.39e ± 0.04
0.44d ± 0.02
acetylpyrazine
1629
A
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.16b ± 0.01
0.22a ± 0.02
1077
A
0.00f ± 0.00 0.32e ± 0.01 0.53d ± 0.02 0.00f ± 0.00
1.38b ± 0.01
0.72c ± 0.02
1.71a ± 0.03
0.77c ± 0.06
pyrazines
thioethers dimethyl disulfide
34
dimethyl trisulfide
1358
B
0.00h ± 0.00 0.25g ± 0.01 0.29e ± 0.01 0.63c ± 0.01
0.71b ± 0.01
0.29f ± 0.02
0.78a ± 0.02
0.41d ± 0.01
furfuryl methyl disulfide
1066
B
0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00
0.96a ± 0.01
0.26b ± 0.01
0.12d ± 0.02
0.16c ± 0.01
hexanal
1083
B
0.00f ± 0.00 0.00f ± 0.00 0.27e ± 0.01 0.00f ± 0.00
0.47d ± 0.02
0.85b ± 0.01
1.14a ± 0.01
0.62c ± 0.01
heptanal
1182
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.23b ± 0.01
0.39a ± 0.01
0.00c ± 0.00
octanal
1281
C
0.41c ± 0.01 0.00g ± 0.00 0.13f ± 0.01 0.63a ± 0.01
0.00g ± 0.00
0.27e ± 0.05
0.58b ± 0.04
0.36d ± 0.06
nonanal
1382
B
1.80c ± 0.04 1.02g ± 0.02 0.95h ± 0.02 2.45a ± 0.04
1.51d ± 0.06
1.37f ± 0.03
2.18b ± 0.01
1.41e ± 0.01
benzaldehyde
1520 A,B 3.24e ± 0.02 0.00h ± 0.00 0.41g ± 0.01 4.50b ± 0.04
3.78d ± 0.02
5.08a ± 0.03
4.37c ± 0.01
1.36f ± 0.01
AB 0.00g ± 0.00 0.35f ± 0.01 0.44ef ± 0.03 35.53a ± 0.09
15.77b ± 0.15
0.50e ± 0.01 10.05c ± 0.05
6.79d ± 0.03
aldehydes
acids acetic acid
1448
butanoic acid
1631
B
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 1.22a ± 0.10
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
benzoic acid
1644
C
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.10a ± 0.01
0.00b ± 0.00
0.00b ± 0.00
octanoic acid
2062 A,B 0.00g ± 0.00 0.95e ± 0.01 1.07e ± 0.05 2.80a ± 0.04
2.01c ± 0.04
1.42d ± 0.03
0.53f ± 0.01
2.31b ± 0.08
nonanoic acid
2171
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.22a ± 0.01
0.00b ± 0.00
0.00b ± 0.00
decanoic acid
2276
C
0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00 2.27a ± 0.09
1.22b ± 0.01
0.69c ± 0.01
0.44d ± 0.01
1.21b ± 0.04
acetic acid ethenyl ester
981
B
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.51a ± 0.01
0.00b ± 0.00
3-methylbutyl acetate
1122
B
0.00e ± 0.00 0.02d ± 0.01 0.03c ± 0.01 0.00e ± 0.00
0.00e ± 0.00
0.06b ± 0.01
0.06b ± 0.01
0.55a ± 0.04
terpinyl acetate
1693
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.06a ± 0.01
0.00b ± 0.00
0.00b ± 0.00
furfuryl acetate
1528
A
0.00f ± 0.00 0.13e ± 0.01 0.22c ± 0.01 0.37a ± 0.01
0.37a ± 0.01
0.28b ± 0.01
0.22d ± 0.02
0.22d ± 0.02
A,B 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
1.39a ± 0.01
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00g ± 0.00 1.57f ± 0.02 1.84e ± 0.04 3.11d ± 0.04
5.67b ± 0.01
3.33c ± 0.01
1.54f ± 0.01
10.79a ± 0.04
esters
alcohols 1-hexanol
1340
linalool
1525
B
35
1547
C
0.34c ± 0.01 0.63b ± 0.01 1.13a ± 0.02 0.00f ± 0.00
0.60b ± 0.01
0.23d ± 0.01
0.14e ± 0.01
0.22d ± 0.01
(2E)-3,7-dimethyl-2,6-octadien-1-ol 1844
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.16a ± 0.01
benzyl alcohol
1880
A
0.00f ± 0.00 0.07f ± 0.01 0.09d ± 0.01 0.46b ± 0.02
0.55a ± 0.01
0.10d ± 0.01
0.11d ± 0.03
0.25c ± 0.01
phenylethyl alcohol
1920
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.60a ± 0.01
0.31b ± 0.01
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
1-butanol
1154
A
0.00b ± 0.00 0.00b ± 0.00 1.26a ± 0.01 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
1-pentanol
1246
B
0.00b ± 0.00 0.00b ± 0.00 0.21a ± 0.01 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
1-heptanol
1451
A
0.20b ± 0.01 0.00c ± 0.00 1.08a ± 0.06 0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
α-terpineol
1698
B
0.00b ± 0.00 0.21a ± 0.01 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
phenol
2020
B
0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00 0.11ab ± 0.01
0.12a ± 0.01
0.06c ± 0.01 0.11ab ± 0.01 0.11b ± 0.01
2-butanone
917
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
3.80a ± 0.02
2.83b ± 0.01
0.00c ± 0.00
2-pentanone
980
B
0.00d ± 0.00 0.06c ± 0.01 0.07b ± 0.01 0.00d ± 0.00
0.00d ± 0.00
0.80a ± 0.02
0.00d ± 0.00
0.00d ± 0.00
2,3-pentanedione
1061
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.26a ± 0.02
0.15b ± 0.01
0.00c ± 0.00
3-penten-2-one
1132
B
0.00e ± 0.00 0.10d ± 0.01 0.22c ± 0.02 0.00e ± 0.00
0.00e ± 0.00
0.256b ± 0.01 0.50a ± 0.01
0.00e ± 0.00
4-methyl-3-penten-2-one
1134
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
1.33b ± 0.04
0.00c ± 0.00
0.00c ± 0.00
6.33a ± 0.12
2-heptanone
1172
B
0.00f ± 0.00 0.06e ± 0.01 0.09d ± 0.01 0.00f ± 0.00
0.13c ± 0.01
0.22b ± 0.01
0.11d ± 0.01
0.88a ± 0.02
2-octanone
1272
B
0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.11b ± 0.01
0.11b ± 0.01
0.21a ± 0.01
2-nonanone
1378
B
0.21b ± 0.01 0.06d ± 0.01 0.07d ± 0.01 0.00e ± 0.00
0.00e ± 0.00
0.11c ± 0.01
0.00e ± 0.00
0.25a ± 0.01
2-nonen-4-one
1476
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.15a ± 0.01
1-phenylethanone
1651
A
0.00d ± 0.00 0.85a ± 0.02 0.56b ± 0.01 0.27c ± 0.01
0.00d ± 0.00
0.00d ± 0.00
0.00d ± 0.00
0.00d ± 0.00
6-methyl-5-hepten-2-one
1300
B
0.00b ± 0.00 0.07a ± 0.01 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
1-octanol
ketones
alkenes
36
β-myrcene
1141
B
0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00 0.00e ± 0.00
0.55b ± 0.01
0.37c ± 0.01
0.19d ± 0.01
2.68a ± 0.21
δ-3-carene
1145
A
0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00
0.18c ± 0.01
1.42a ± 0.02
0.90b ± 0.01
0.00d ± 0.00
dl-limonene
1166
B
0.00f ± 0.00 0.34e ± 0.01 0.53d ± 0.01 0.54d ± 0.01
0.36e ± 0.01
1.81b ± 0.02
1.60c ± 0.01
2.23a ± 0.01
trans-β-ocimene
1213
A
0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00 0.00d ± 0.00
0.66b ± 0.01
0.11c ± 0.01
0.00d ± 0.00
0.81a ± 0.01
γ-terpinene
1230
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.25a ± 0.01
D-limonene
1196
B
1.12a ± 0.03 0.34b ± 0.01 0.00c ± 0.00 0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
p-cymene
1256
A
0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00 0.00b ± 0.00
0.45a ± 0.01
limonene
1188
B
0.00b ± 0.00 0.00b ± 0.00 1.66a ± 0.01 0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
0.00b ± 0.00
styrene
1237
B
0.00c ± 0.00 0.21b ± 0.01 1.03a ± 0.01 0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
0.00c ± 0.00
x
Kovats indices of compounds on HP - INNOWAX column.
y
Identification method of the compounds: (A) mass spectrum and Kovats index according to literature (Niu et al., 2011; Xiao et al., 2011; Xiao et al.,
2014); (B) mass spectrum compared with databases； （C）mass spectrum and Kovats index agree with that of the authentic compound run under the same conditions of the samples. z
Results were expressed as mean value ± standard deviation (n = 2).
Values bearing different lowercase letters (a, b, c, d and e) were significantly different (p ≤ 0.05).
37
Highlights A procedure was set up to produce extract from beef bone by lipase pretreatment. An excellent beef aroma was formed due to plentiful amino acids in the extract. Industrial beef by-product was used to produce food flavouring by Maillard reaction.
38