Research Article Received: 6 July 2013
Revised: 31 October 2013
Accepted article published: 8 November 2013
Published online in Wiley Online Library: 4 December 2013
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6472
A novel method for beef potentiator preparation and identification of its characteristic aroma compounds Xianli Gao,a,b∗ Shuang Yan,a,b Bao Yang,c Jian Lua,b∗ and Zhao Jina,b Abstract BACKGROUND: Beef potentiator (BP) is the most popular savoury flavour and regarded as the soul of the modern food industry. In this work, BP was prepared by a novel method with Aspergillus oryzae and Aspergillus niger (BPSF). Three other BPs prepared using commercial enzymes (Protamex, Flavourzyme and papain; BPCEs) were used as controls to investigate its aroma characteristics and related compounds. RESULTS: Sensory evaluation showed that BPSF possessed more favourable and distinctive sauce-like, meat-like, roast and alcoholic attributes when compared with BPCEs. Significantly higher contents (peak areas) and proportions of pyrazines, pyrroles, sulfurous compounds and alcohols in BPSF were responsible for its sensory characteristics, and most of these aroma compounds were derived from microbial metabolism during beef koji preparation and the Maillard reaction. CONCLUSION: BP prepared by synergistic fermentation with A. oryzae and A. niger is a potential alternative for BP preparation. c 2013 Society of Chemical Industry Keywords: beef potentiator; synergistic fermentation; gas chromatography–mass spectrometry (GC-MS); aroma compounds; sensory evaluation
INTRODUCTION
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Savoury flavour is one of the essential condiments in food industry. Its annual production and output value have been more than 1 million tons and 10 billion Chinese Yuan in China, in which BP is the most popular product.1 Undoubtedly, the market demand of BP will increase steadily with the rapid development of food industry in the future. Currently, the manufacturing processes of BP mainly consist of beef cooking, enzymatic hydrolysis, moderate Maillard reaction, drying and flavour enhancement (i.e. chemical beef note, sugar, salt, monosodium glutamate, I + G disodium 5’-inosinate and disodium 5’-guanylate, spices).2 The variety, amount of employed enzymes and the processing conditions during beef hydrolysate preparation were generally regarded as the prerequisites to prepare a favourable BP. The favourable aroma as well as high content of free amino acids and low-molecular-weight peptides of the hydrolysate is crucial to the final product.2 – 5 Therefore, the final BP quality is mostly determined by the aroma of beef hydrolysate. Owing to the high price and usage amount (6–12 g kg−1 ) of commercial enzymes (e.g. Novozymes’ Protamex, approximately 360 RMB kg−1 ) and the lack of natural aroma, BP prepared by the present method is of high production cost and weak flavour.2,6 Meanwhile, the addition of chemical beef note has caused increasing safety concerns in the past several years, especially in the past 2 years in China. Thus it is required to find an effective and economical method to improve the natural aroma and safety and reduce the production cost of BP. It is reported that Aspergillus oryzae has the largest expansion of hydrolytic genes.7 Among the proteases secreted by A. J Sci Food Agric 2014; 94: 1648–1656
oryzae, neutral proteases – endoproteases involved in elevating utilization efficiency of proteins – are the dominant proteases, whereas acid proteases – exoproteases involved in free amino acids released from proteins and peptides – are inadequate.8 By contrast, A. niger secretes large quantities of exoproteases and a small quantity of endoproteases.8 In our previous study, strong soy sauce-like aroma present at the end of A. oryzae koji preparation was observed.9 In addition, A. oryzae and A. niger are affirmed as GRAS (generally recognized as safe) by the Food and Drug Administration and widely used in different biotechnological processes.8,10 Thus mixing A. oryzae with A. niger at an appropriate ratio to prepare beef koji might be a novel and potential approach to produce BP due to the rich and balanced proteases and ‘sauce note’ secreted by the fungi, which not only could replace the commercial enzymes and chemical beef note used in BP but also improve its edible safety. The optimal inoculation ratio (3:1) of A. oryzae and A. niger and manufacturing processes have been established in
∗
Correspondence to: Xianli Gao or Jian Lu, Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. E-mail: [email protected]; [email protected]
a Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China b National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China c South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
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c 2013 Society of Chemical Industry
A novel method for beef potentiator preparation
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our laboratory. The controls (beef potentiators prepared using commercial enzymes, BPCEs) were prepared according to the manufacturing processes of a professional flavour and fragrance company (Guangzhou Ririxiang Food Co., Ltd, Guangzhou, China). Thus the objectives of this work were (i) to evaluate the sensory characteristics of beef potentiator prepared by synergistic fermentation with A.oryzae and A.niger (BPSF) and (ii) to investigate the aroma compounds related to its sensory characteristics when compared with BPCEs.
MATERIALS AND METHODS Materials and chemicals Fresh beef and hexane-defatted soy meal, containing 198.0 and 475.1 g kg−1 proteins, 12.2 and 211.0 g kg−1 carbohydrates, 765.9 and 75.4 g kg−1 moisture, and 11.4 and 48.8 g kg−1 ash, respectively, were purchased from a local supermarket in Wuxi city (Vanguard, Jiangsu, China) and Yuwang Industrial Co., Ltd (Shandong, China). Wheat flour and wheat bran were purchased from a local farmer’s market. Protamex and Flavourzyme were purchased from Novozymes (Beijing, China). Papain was obtained from Baiao Biochemistry Co., Ltd (Guangdong, China). Other chemicals used in this study were of the highest commercial grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). A. oryzae was screened from a soy sauce factory soil and preserved at the China centre for type culture collection (CCTCC NO. M2012265), and A. niger 3.350 was commercially available from Guangdong culture collection centre. Preparation of BPSF and BPCEs Seed koji preparation Wheat bran (20 g), wheat flour (5 g) and deionized water (20 g) were thoroughly mixed and two aliquots of the mixture were sterilized in conical flasks at 121 ◦ C for 30 min. Then the mixtures were cooled to 40 ◦ C and inoculated with three loops of A. oryzae spores and A. niger spores, respectively. The mixtures were then cultured at 30 ◦ C for 65 h and 96 h to obtain two kinds of seed kojis. The resulting seed kojis were kept in closure pockets in a refrigerator (4 ◦ C), respectively. Beef koji preparation (1) One kilogram of fresh beef was minced and then blended with a mixture of 150 g wheat bran, 50 g hexane-defatted soy meal
and 160 g deionized water. (2) The above mixture was autoclaved at 121 ◦ C for 20 min. (3) three hundred grams of wheat flour were inoculated with spores of A. oryzae (3.0 × 107 spores g−1 of the resulting mixture) and A. niger (1.0 × 107 spores g−1 of the resulting mixture) , which was further mixed with the above cooled mixture (below 40 ◦ C). (4) The resulting mixture was then cultured at 28 ◦ C for 46 h and the obtained koji was kept in a refrigerator (4 ◦ C) for further use. The protease activities of beef koji and three commercial enzymes (Protamex, Flavourzyme, papain) were assayed by the method of Gao et al.8 Preparation of BPSF The pH of a mixture of pulverized beef koji and deionized water (beef koji:deionized water = 1:3, w/w) was adjusted to 7.0 with 2 mol L−1 NaOH solution; then the mixture was slowly stirred in a sealed three-neck round-bottom flask at 45 ◦ C for 10 h. At the end of the hydrolysis, the liquid was autoclaved at 121 ◦ C for 60 min in a sealed stainless container and then centrifuged at 2070 × g for 20 min. Finally, the supernatant was kept in a refrigerator (4 ◦ C) for further analysis. Preparation of BPCEs The pretreatment methods and quantities of raw materials employed to BPCEs were the same as BPSF except for no spores inoculated in steamed substrates. Three commercial enzymes (Protamex, Flavourzyme and papain) were used to prepare BPCEs, and the varieties, amounts and application conditions of commercial enzymes used in this study were in accordance with the production processes of traditional BP. Details of the commercial enzymes and parameters of manufacturing processes used in BPCEs preparation are listed in Table 1. Sensory evaluation Sensory evaluation training Descriptive analysis (DA) was performed to investigate the differences in aroma between BPSF and BPCEs.11 Analysis was carried out with a panel of nine flavourists (six men and three women, aged 24–46) in a professional flavour and fragrance company (Guangzhou Ririxiang Food Co., Ltd, Guangzhou, China). Five training sessions (1 h per session) were held within a week. The panellists were subjected to a ranking test with a series of seven suprathreshold aqueous solutions (25 mL in Teflon vials) of acetic acid (sour), ethanol (alcoholic), ammonia
Table 1. Information of commercial enzymes and parameters of manufacturing processes used in BP preparation Protease (U g−1 ) Sample namea BPSF BPPTF BPPPF BPPT
Name Beef koji Protamex Flavourzyme Papain Flavourzyme Protamex
Protease activity /substrate (U g−1 )
Activityb — 131 830N 33 620N 86 500N 33 620N 131 830N
— ndA 384A 794A 384A ndA
876.0N 791.0N 100.9N 519.0N 100.9N 791.0N
407.0A ndA 1.2A 4.8A 1.2A ndA
Temperature (◦ C)
Time (h)
45 50
10 10
50
10
50
10
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a BPTPF, BPPPF, BPPT are BPs prepared using Protamex (6/1000, Protamex/substrate, w/w) and Flavourzyme (3/1000, Flavourzyme/substrate, w/w), papain (6/1000, papain /substrate, w/w) and Flavourzyme (3/1000, Flavourzyme/substrate, w/w) and Protamex (6/1000, Protamex/substrate, w/w), respectively. b —, no commercial enzyme was used. N neutral protease; A acid protease; nd, not detected.
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X Gao et al.
(ammonia), trimethylpyrazine (roast), trimethylamine (fishy) and bis(2-methyl-3-furyl) disulfide (meat-like), and then were asked to score the aroma intensities on a linear scale from 0 to 9. Meanwhile, the panellists were also asked to rank the sauce-like intensity of 10 g L−1 soy sauce solution (soy sauce, Haday, Foshan City, Guangdong, China) on a linear scale from 0 to 9. Sensory evaluation was performed in a sensory panel room at 23 ± 2 ◦ C at three different sessions. Results of sensory evaluation were discussed and agreement eventually obtained by all panellists. Sensory evaluation All samples (10 mL) were coded with random three-digit numbers, and served to the panellists in a randomized complete block design. The seven aroma intensities of samples were evaluated using a linear scale from 0 (extremely weak) to 9 (extremely strong). Finally, the results – intensities of the seven aroma attributes – were plotted in a spider web diagram. Aroma compounds analysis Aroma compounds collection by solid-phase microextraction (SPME) An SPME fibre coated with 75 µm carboxen–polydimethylsiloxane (Supelco, Bellefonte, PA, USA) was selected to collect aroma compounds for its high sensitivity and good selectivity to polar and non-polar compounds.12 Before sampling, the fibre was preconditioned for 1 h 30 min at 275 and 250 ◦ C, respectively, in the gas chromatograph injector port to eliminate possible residues on the coated fibre. Samples (5 mL) saturated with NaCl were sealed in a dedicated bottle and preheated at 50 ◦ C, stirred by a magnetic stirring bar. The adsorption time was 40 min, and the concentrates were desorbed at 250 ◦ C in the injection port of the gas chromatograph (Finnigan Trace GC-2000, Austin, TX, USA) by holding in the splitless mode for 5 min. The SPME was cleaned by keeping it in the injection port for an additional 5 min. Gas chromatography–mass spectrometry (GC-MS) GC-MS analysis was carried out using a Finnigan TRACE GC-2000 gas chromatography–mass spectrometer (Finnigan, Austin, TX, USA), equipped with a DB-Wax column (30 m length × 0.25 mm i.d × 0.25 µm film thickness; J&W Scientific, Folsom, CA, USA). Aroma compounds adsorbed on the fibres were transferred into the gas chromatograph injector in splitless mode with an injection purge-off time of 5 min and injection temperature of 250 ◦ C. The initial temperature of the gas chromatograph oven was held for 4 min at 40 ◦ C, then raised to 60 ◦ C at 5 ◦ C min−1 , and finally increased to 230 ◦ C (held for 9 min) at 12 ◦ C min−1 . Ultra-highpurity helium was used as carrier gas at a constant speed of 0.8 mL min−1 . Mass spectrometer conditions were as follows: MSD capillary direct-interface temperature 250 ◦ C; ionization energy 70 eV; mass range 30–450 a.m.u.
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Identification and quantification of the aroma compounds Each compound was tentatively identified by comparing its mass spectral data with those of a NIST library (including Wiley and Mainlib) and published literature, and finally confirmed by the Kovats retention index (RI).2 – 4,9,12 – 19 The RIs based on DB-Wax column were calculated using C10 –C33 n-alkanes as standards. Quantitative analysis was based on the ratio between the peak area of a particular compound and the total peak area of all compounds in a sample.
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Figure 1. Results of sensory evaluation of BPSF and BPCEs.
Statistical analysis All determinations were conducted in triplicate. The results were subjected to one-way analysis of variance (ANOVA). Duncan’s new multiple range test was performed to determine the significant difference between samples within 95% confidence intervals using SPSS 15.0 software (SPSS Inc, Chicago, IL, USA).
RESULTS AND DISCUSSION Sensory evaluation The aroma profiles of BPSF and BPCEs based on descriptive analysis tests by the nine panellists are shown in Fig. 1. BPSF showed a more favourable and distinctive complex of sauce-like, meat-like, roast and alcoholic aromas compared with BPCEs. ANOVA revealed that BPSF possessed significantly higher intensities of saucelike, roast and alcoholic aromas than those of BPCEs (P < 0.05). Meanwhile, the four samples all possessed quite low intensities of sour, ammonia and fishy, and no significant differences among them was observed. The different aroma profiles between BPSF and BPCEs suggested that the aroma compounds and/or their percentages in the four BPs might be different. Aroma compounds detected by SPME-GC-MS To clarify the reasons for the different aroma profiles of BPSF and BPCEs, aroma compounds in BPSF and BPCEs were identified and the results are summarized in Tables 2 and 3. In total, 79, 76, 64 and 67 aroma compounds were identified in BPSF, BPPTF, BPPPF and BPPT, respectively, and only 41 aroma compounds were common in the four samples. Aldehydes and ketones (24–30 compounds, 43.21–79.56%), pyrazines (5–14 compounds, 1.81–35.18%), sulfurous compounds (6–10 compounds, 2.81–3.47%) and furan(one)s (8–12 compounds, 7.62–30.37%) constituted the main portion of aroma compounds in BPSF and BPCEs. Results of previous research showed that 70 volatile and 48 odour-active compounds were detected in two kinds of beef flavours, respectively.3,4 Noticeably, sulfurous compounds and nitrogen-containing compounds accounted for the main portion of 48 odour-active compounds, while only 16 of them were also identified in this work.4 Therefore, it was reasonable to infer that the aroma characteristics of BPSF were different from the
c 2013 Society of Chemical Industry
J Sci Food Agric 2014; 94: 1648–1656
A novel method for beef potentiator preparation
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Table 2. Peak areas (contents) of different kinds of aroma compounds detected by SPME-GC-MS in BPSF and BPCEs Peak area (× 106 ) BPSF Acids Alcohols Aldehydes and ketones Esters Furan(one)s Phenols Pyrazines Pyrroles Sulfurous compounds Total peak area
3.81 41.59 239.77 5.62 42.25 1.87 195.22 5.75 19.21 555.09
± ± ± ± ± ± ± ± ± ±
0.45a 3.58a 23.63a 0.59a 4.21c 0.12a 17.99a 0.44a 1.79a 52.12a
BPPTF 2.22 4.91 231.11 4.80 84.95 2.03 23.60 0.73 10.62 364.97
± ± ± ± ± ± ± ± ± ±
0.38b 0.61b 21.62ab 0.51b 8.06b 0.15a 2.20b 0.08b 1.18b 34.12b
BPPPF 0.92 3.38 117.79 2.10 11.76 0.36 6.69 0.87 4.17 148.04
± ± ± ± ± ± ± ± ± ±
0.16c 0.52b 10.93c 0.17d 1.40d 0.05b 0.69bc 0.09b 0.45c 14.23c
BPPT 3.21 6.78 202.69 3.59 101.69 0.15 6.10 0.32 10.33 334.86
± ± ± ± ± ± ± ± ± ±
0.27a 0.86b 18.40b 0.34c 8.40a 0.03c 0.50c 0.06c 1.05b 29.02b
Values are expressed as means ± standard deviations (n = 3); values in the same row followed by different letters are significantly different (P < 0.05).
above beef flavours due to their obvious differences in aroma compounds and percentages. The peak areas of different kinds of aroma compounds in BPSF and BPCEs are shown in Table 2. The peak areas of alcohols, esters, pyrazines, pyrroles, sulfurous compounds and the total peak area in BPSF were significantly higher than those in BPCEs (P < 0.05). Although the percentages of aldehydes and ketones in BPPTF, BPPPF and BPPT were about 63%, 80% and 61%, respectively, which were higher than that in BPSF, the peak area of aldehydes and ketones in BPSF was higher than that in BPCEs. The above results indicated that BPSF produced higher contents of aroma compounds, especially pyrazines and sulfurous compounds. To understand the aroma differences between BPSF and BPCEs, it was important to investigate the origins of the aroma compounds. The combination of lipid oxidation, Maillard reaction and microbial activities should be responsible for the aroma characteristics of BPSF.
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Aroma compounds from lipid oxidation Aroma compounds derived from lipid oxidation, including aliphatic aldehydes (hexanal, tetradecanal), certain ketones (3-penten-2-one, 2,3-heptanedione, 2-heptanone, 2,3octanedione, 2,6-dimethyl-3-heptanone, 2-octanone), aliphatic alcohols (2-propen-1-ol, 1-penten-3-ol, 1-hexanol, 1-octen-3-ol, 2-ethylhexanol, 1-octanol, dodecyl alcohol) and aliphatic acids (hexanoic acid, octanoic acid, tetradecanoic acid), were detected in this work. In addition, some long-chain acids and alcohols in esters were also derived from products of lipid oxidation, such as 1-hexanol in hexyl acetate, hexanoic acid in ethyl hexanoate and hexadecanoic acid in ethyl hexadecanoate. 2-Alkylfurans formed from the corresponding α,β-unsaturated aldehydes were also lipid derived.20 Most of the above aroma compounds had great relevance to the characteristic aroma since lipid oxidation products were confirmed to play an important role in the sapid and aromatic characteristics of BP.2 In this work, although a total of five acids (Table 3) were detected in four BPs, only two acids (butyric acid and tetradecanoic acid) were detected in BPSF. However, sensory evaluation indicated that BPSF possessed a stronger sour attribute than BPCEs, even though the latter contained more varieties and higher percentages of acids. The lower threshold and higher content (peak area, Table 2) of butyric acid and some acids (i.e. acetic acid) undetected in this work due to the limitation of SPME-GC-MS might be responsible for
this phenomenon. The occurrence of acetates in Table 3 confirmed the existence of acetic acid in BPSF and BPCEs. As shown in Tables 2 and 3, the peak area (content) and percentage of alcohols in BPSF were about 8.5- and 5.6, 12.3- and 3.3-, 6.1- and 3.7-fold of those in BPPTF, BPPPF and BPPT, respectively. In particular, the percentage of ethanol in BPSF was about 4.4-, 3.7- and 3.6-fold of that in BPPTF, BPPPF and BPPT, respectively. The results were consistent with the sensory evaluation result concerning alcoholic attribute (Fig. 1). In addition, 1-octen-3-ol, 2-ethylhexanol and phenethyl alcohol – aroma-active compounds in beef flavour2 and soy sauce11,14 – were detected in BPSF with much higher percentages. These compounds, especially 1-octen-3-ol, can show a strong modification to meat aroma or intensify the perception of samples associated with ‘beefy’ and ‘meaty’ aromas.2 As shown in Table 3, 28, 30, 24 and 27 aldehyde and ketone aroma compounds were detected, in which BPSF possessed the lowest percentage of aldehydes and ketones when compared with BPCEs. Individually, 2,6-dimethyl-3-heptanone (7.14%), 2-phenyl-2-butenal (4.47%), 3-methylbutanal (4.46%), acetone (3.93%) and 2-methylbutanal (3.81%) were the most abundant compounds of aldehydes and ketones in BPSF, whereas 3-methylbutanal (13.07–22.24%), benzaldehyde (3.73–9.68%), 2-methylbutanal (2.67–9.57%), acetone (3.60–9.51%) and 2butanone (3.95–9.18%) constituted the majority of aldehydes and ketones in BPCEs. Most of the above aroma compounds, such as 3-methylbutanal, 2-methylbutanal and benzaldehyde, were aroma-active compounds in beef flavour2 and soy sauce.11,14 Thus the discrepancies in percentages and compositions of aldehydes and ketones between BPSF and BPCEs would lead to their differences in aroma profiles to some extent. As shown in Table 3, only 2-methylfuran (0.27%) and 2phenylfuran (0.26%) were detected in BPSF. However, there were more varieties and a higher proportion of 2-alkylfurans, such as 2-ethylfuran (0.15–0.27%), 2-pentylfuran (0.12–0.27%) and 2phenylfuran (0.10–2.24%), detected in BPCEs. The differences in 2-alkylfuran compositions of BPSF and BPCEs might partly result in their aroma differences, such as their roast, burnt and meaty attributes.2,20 In this work, only four esters with low total peak area (5.62 × 106 ) and percentage (1.03%) were detected in BPSF. Similar results were also observed in BPCEs and previous studies.2,4 Esters, especially ethyl esters, in food could contribute a good flavour by minimizing
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X Gao et al.
Table 3. Aroma compounds and their percentages detected by SPME-GC-MS in BPSF and BPCEs Percentages of peak areas (%) Compounds
RIa
IDb
Acids Butyric acid Hexanoic acid Octanoic acid 2-Hydroxycinnamic acid Tetradecanoic acid Total (5)
1607 1823 2026 2324 2688
A,B A,B A,B A A,B
Rancid/sweaty Sour/pungent Sweaty, sour, grassy — Waxy, oily
Revised: 31 October 2013
Accepted article published: 8 November 2013
Published online in Wiley Online Library: 4 December 2013
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6472
A novel method for beef potentiator preparation and identification of its characteristic aroma compounds Xianli Gao,a,b∗ Shuang Yan,a,b Bao Yang,c Jian Lua,b∗ and Zhao Jina,b Abstract BACKGROUND: Beef potentiator (BP) is the most popular savoury flavour and regarded as the soul of the modern food industry. In this work, BP was prepared by a novel method with Aspergillus oryzae and Aspergillus niger (BPSF). Three other BPs prepared using commercial enzymes (Protamex, Flavourzyme and papain; BPCEs) were used as controls to investigate its aroma characteristics and related compounds. RESULTS: Sensory evaluation showed that BPSF possessed more favourable and distinctive sauce-like, meat-like, roast and alcoholic attributes when compared with BPCEs. Significantly higher contents (peak areas) and proportions of pyrazines, pyrroles, sulfurous compounds and alcohols in BPSF were responsible for its sensory characteristics, and most of these aroma compounds were derived from microbial metabolism during beef koji preparation and the Maillard reaction. CONCLUSION: BP prepared by synergistic fermentation with A. oryzae and A. niger is a potential alternative for BP preparation. c 2013 Society of Chemical Industry Keywords: beef potentiator; synergistic fermentation; gas chromatography–mass spectrometry (GC-MS); aroma compounds; sensory evaluation
INTRODUCTION
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Savoury flavour is one of the essential condiments in food industry. Its annual production and output value have been more than 1 million tons and 10 billion Chinese Yuan in China, in which BP is the most popular product.1 Undoubtedly, the market demand of BP will increase steadily with the rapid development of food industry in the future. Currently, the manufacturing processes of BP mainly consist of beef cooking, enzymatic hydrolysis, moderate Maillard reaction, drying and flavour enhancement (i.e. chemical beef note, sugar, salt, monosodium glutamate, I + G disodium 5’-inosinate and disodium 5’-guanylate, spices).2 The variety, amount of employed enzymes and the processing conditions during beef hydrolysate preparation were generally regarded as the prerequisites to prepare a favourable BP. The favourable aroma as well as high content of free amino acids and low-molecular-weight peptides of the hydrolysate is crucial to the final product.2 – 5 Therefore, the final BP quality is mostly determined by the aroma of beef hydrolysate. Owing to the high price and usage amount (6–12 g kg−1 ) of commercial enzymes (e.g. Novozymes’ Protamex, approximately 360 RMB kg−1 ) and the lack of natural aroma, BP prepared by the present method is of high production cost and weak flavour.2,6 Meanwhile, the addition of chemical beef note has caused increasing safety concerns in the past several years, especially in the past 2 years in China. Thus it is required to find an effective and economical method to improve the natural aroma and safety and reduce the production cost of BP. It is reported that Aspergillus oryzae has the largest expansion of hydrolytic genes.7 Among the proteases secreted by A. J Sci Food Agric 2014; 94: 1648–1656
oryzae, neutral proteases – endoproteases involved in elevating utilization efficiency of proteins – are the dominant proteases, whereas acid proteases – exoproteases involved in free amino acids released from proteins and peptides – are inadequate.8 By contrast, A. niger secretes large quantities of exoproteases and a small quantity of endoproteases.8 In our previous study, strong soy sauce-like aroma present at the end of A. oryzae koji preparation was observed.9 In addition, A. oryzae and A. niger are affirmed as GRAS (generally recognized as safe) by the Food and Drug Administration and widely used in different biotechnological processes.8,10 Thus mixing A. oryzae with A. niger at an appropriate ratio to prepare beef koji might be a novel and potential approach to produce BP due to the rich and balanced proteases and ‘sauce note’ secreted by the fungi, which not only could replace the commercial enzymes and chemical beef note used in BP but also improve its edible safety. The optimal inoculation ratio (3:1) of A. oryzae and A. niger and manufacturing processes have been established in
∗
Correspondence to: Xianli Gao or Jian Lu, Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. E-mail: [email protected]; [email protected]
a Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China b National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China c South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
www.soci.org
c 2013 Society of Chemical Industry
A novel method for beef potentiator preparation
www.soci.org
our laboratory. The controls (beef potentiators prepared using commercial enzymes, BPCEs) were prepared according to the manufacturing processes of a professional flavour and fragrance company (Guangzhou Ririxiang Food Co., Ltd, Guangzhou, China). Thus the objectives of this work were (i) to evaluate the sensory characteristics of beef potentiator prepared by synergistic fermentation with A.oryzae and A.niger (BPSF) and (ii) to investigate the aroma compounds related to its sensory characteristics when compared with BPCEs.
MATERIALS AND METHODS Materials and chemicals Fresh beef and hexane-defatted soy meal, containing 198.0 and 475.1 g kg−1 proteins, 12.2 and 211.0 g kg−1 carbohydrates, 765.9 and 75.4 g kg−1 moisture, and 11.4 and 48.8 g kg−1 ash, respectively, were purchased from a local supermarket in Wuxi city (Vanguard, Jiangsu, China) and Yuwang Industrial Co., Ltd (Shandong, China). Wheat flour and wheat bran were purchased from a local farmer’s market. Protamex and Flavourzyme were purchased from Novozymes (Beijing, China). Papain was obtained from Baiao Biochemistry Co., Ltd (Guangdong, China). Other chemicals used in this study were of the highest commercial grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). A. oryzae was screened from a soy sauce factory soil and preserved at the China centre for type culture collection (CCTCC NO. M2012265), and A. niger 3.350 was commercially available from Guangdong culture collection centre. Preparation of BPSF and BPCEs Seed koji preparation Wheat bran (20 g), wheat flour (5 g) and deionized water (20 g) were thoroughly mixed and two aliquots of the mixture were sterilized in conical flasks at 121 ◦ C for 30 min. Then the mixtures were cooled to 40 ◦ C and inoculated with three loops of A. oryzae spores and A. niger spores, respectively. The mixtures were then cultured at 30 ◦ C for 65 h and 96 h to obtain two kinds of seed kojis. The resulting seed kojis were kept in closure pockets in a refrigerator (4 ◦ C), respectively. Beef koji preparation (1) One kilogram of fresh beef was minced and then blended with a mixture of 150 g wheat bran, 50 g hexane-defatted soy meal
and 160 g deionized water. (2) The above mixture was autoclaved at 121 ◦ C for 20 min. (3) three hundred grams of wheat flour were inoculated with spores of A. oryzae (3.0 × 107 spores g−1 of the resulting mixture) and A. niger (1.0 × 107 spores g−1 of the resulting mixture) , which was further mixed with the above cooled mixture (below 40 ◦ C). (4) The resulting mixture was then cultured at 28 ◦ C for 46 h and the obtained koji was kept in a refrigerator (4 ◦ C) for further use. The protease activities of beef koji and three commercial enzymes (Protamex, Flavourzyme, papain) were assayed by the method of Gao et al.8 Preparation of BPSF The pH of a mixture of pulverized beef koji and deionized water (beef koji:deionized water = 1:3, w/w) was adjusted to 7.0 with 2 mol L−1 NaOH solution; then the mixture was slowly stirred in a sealed three-neck round-bottom flask at 45 ◦ C for 10 h. At the end of the hydrolysis, the liquid was autoclaved at 121 ◦ C for 60 min in a sealed stainless container and then centrifuged at 2070 × g for 20 min. Finally, the supernatant was kept in a refrigerator (4 ◦ C) for further analysis. Preparation of BPCEs The pretreatment methods and quantities of raw materials employed to BPCEs were the same as BPSF except for no spores inoculated in steamed substrates. Three commercial enzymes (Protamex, Flavourzyme and papain) were used to prepare BPCEs, and the varieties, amounts and application conditions of commercial enzymes used in this study were in accordance with the production processes of traditional BP. Details of the commercial enzymes and parameters of manufacturing processes used in BPCEs preparation are listed in Table 1. Sensory evaluation Sensory evaluation training Descriptive analysis (DA) was performed to investigate the differences in aroma between BPSF and BPCEs.11 Analysis was carried out with a panel of nine flavourists (six men and three women, aged 24–46) in a professional flavour and fragrance company (Guangzhou Ririxiang Food Co., Ltd, Guangzhou, China). Five training sessions (1 h per session) were held within a week. The panellists were subjected to a ranking test with a series of seven suprathreshold aqueous solutions (25 mL in Teflon vials) of acetic acid (sour), ethanol (alcoholic), ammonia
Table 1. Information of commercial enzymes and parameters of manufacturing processes used in BP preparation Protease (U g−1 ) Sample namea BPSF BPPTF BPPPF BPPT
Name Beef koji Protamex Flavourzyme Papain Flavourzyme Protamex
Protease activity /substrate (U g−1 )
Activityb — 131 830N 33 620N 86 500N 33 620N 131 830N
— ndA 384A 794A 384A ndA
876.0N 791.0N 100.9N 519.0N 100.9N 791.0N
407.0A ndA 1.2A 4.8A 1.2A ndA
Temperature (◦ C)
Time (h)
45 50
10 10
50
10
50
10
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a BPTPF, BPPPF, BPPT are BPs prepared using Protamex (6/1000, Protamex/substrate, w/w) and Flavourzyme (3/1000, Flavourzyme/substrate, w/w), papain (6/1000, papain /substrate, w/w) and Flavourzyme (3/1000, Flavourzyme/substrate, w/w) and Protamex (6/1000, Protamex/substrate, w/w), respectively. b —, no commercial enzyme was used. N neutral protease; A acid protease; nd, not detected.
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(ammonia), trimethylpyrazine (roast), trimethylamine (fishy) and bis(2-methyl-3-furyl) disulfide (meat-like), and then were asked to score the aroma intensities on a linear scale from 0 to 9. Meanwhile, the panellists were also asked to rank the sauce-like intensity of 10 g L−1 soy sauce solution (soy sauce, Haday, Foshan City, Guangdong, China) on a linear scale from 0 to 9. Sensory evaluation was performed in a sensory panel room at 23 ± 2 ◦ C at three different sessions. Results of sensory evaluation were discussed and agreement eventually obtained by all panellists. Sensory evaluation All samples (10 mL) were coded with random three-digit numbers, and served to the panellists in a randomized complete block design. The seven aroma intensities of samples were evaluated using a linear scale from 0 (extremely weak) to 9 (extremely strong). Finally, the results – intensities of the seven aroma attributes – were plotted in a spider web diagram. Aroma compounds analysis Aroma compounds collection by solid-phase microextraction (SPME) An SPME fibre coated with 75 µm carboxen–polydimethylsiloxane (Supelco, Bellefonte, PA, USA) was selected to collect aroma compounds for its high sensitivity and good selectivity to polar and non-polar compounds.12 Before sampling, the fibre was preconditioned for 1 h 30 min at 275 and 250 ◦ C, respectively, in the gas chromatograph injector port to eliminate possible residues on the coated fibre. Samples (5 mL) saturated with NaCl were sealed in a dedicated bottle and preheated at 50 ◦ C, stirred by a magnetic stirring bar. The adsorption time was 40 min, and the concentrates were desorbed at 250 ◦ C in the injection port of the gas chromatograph (Finnigan Trace GC-2000, Austin, TX, USA) by holding in the splitless mode for 5 min. The SPME was cleaned by keeping it in the injection port for an additional 5 min. Gas chromatography–mass spectrometry (GC-MS) GC-MS analysis was carried out using a Finnigan TRACE GC-2000 gas chromatography–mass spectrometer (Finnigan, Austin, TX, USA), equipped with a DB-Wax column (30 m length × 0.25 mm i.d × 0.25 µm film thickness; J&W Scientific, Folsom, CA, USA). Aroma compounds adsorbed on the fibres were transferred into the gas chromatograph injector in splitless mode with an injection purge-off time of 5 min and injection temperature of 250 ◦ C. The initial temperature of the gas chromatograph oven was held for 4 min at 40 ◦ C, then raised to 60 ◦ C at 5 ◦ C min−1 , and finally increased to 230 ◦ C (held for 9 min) at 12 ◦ C min−1 . Ultra-highpurity helium was used as carrier gas at a constant speed of 0.8 mL min−1 . Mass spectrometer conditions were as follows: MSD capillary direct-interface temperature 250 ◦ C; ionization energy 70 eV; mass range 30–450 a.m.u.
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Identification and quantification of the aroma compounds Each compound was tentatively identified by comparing its mass spectral data with those of a NIST library (including Wiley and Mainlib) and published literature, and finally confirmed by the Kovats retention index (RI).2 – 4,9,12 – 19 The RIs based on DB-Wax column were calculated using C10 –C33 n-alkanes as standards. Quantitative analysis was based on the ratio between the peak area of a particular compound and the total peak area of all compounds in a sample.
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Figure 1. Results of sensory evaluation of BPSF and BPCEs.
Statistical analysis All determinations were conducted in triplicate. The results were subjected to one-way analysis of variance (ANOVA). Duncan’s new multiple range test was performed to determine the significant difference between samples within 95% confidence intervals using SPSS 15.0 software (SPSS Inc, Chicago, IL, USA).
RESULTS AND DISCUSSION Sensory evaluation The aroma profiles of BPSF and BPCEs based on descriptive analysis tests by the nine panellists are shown in Fig. 1. BPSF showed a more favourable and distinctive complex of sauce-like, meat-like, roast and alcoholic aromas compared with BPCEs. ANOVA revealed that BPSF possessed significantly higher intensities of saucelike, roast and alcoholic aromas than those of BPCEs (P < 0.05). Meanwhile, the four samples all possessed quite low intensities of sour, ammonia and fishy, and no significant differences among them was observed. The different aroma profiles between BPSF and BPCEs suggested that the aroma compounds and/or their percentages in the four BPs might be different. Aroma compounds detected by SPME-GC-MS To clarify the reasons for the different aroma profiles of BPSF and BPCEs, aroma compounds in BPSF and BPCEs were identified and the results are summarized in Tables 2 and 3. In total, 79, 76, 64 and 67 aroma compounds were identified in BPSF, BPPTF, BPPPF and BPPT, respectively, and only 41 aroma compounds were common in the four samples. Aldehydes and ketones (24–30 compounds, 43.21–79.56%), pyrazines (5–14 compounds, 1.81–35.18%), sulfurous compounds (6–10 compounds, 2.81–3.47%) and furan(one)s (8–12 compounds, 7.62–30.37%) constituted the main portion of aroma compounds in BPSF and BPCEs. Results of previous research showed that 70 volatile and 48 odour-active compounds were detected in two kinds of beef flavours, respectively.3,4 Noticeably, sulfurous compounds and nitrogen-containing compounds accounted for the main portion of 48 odour-active compounds, while only 16 of them were also identified in this work.4 Therefore, it was reasonable to infer that the aroma characteristics of BPSF were different from the
c 2013 Society of Chemical Industry
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A novel method for beef potentiator preparation
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Table 2. Peak areas (contents) of different kinds of aroma compounds detected by SPME-GC-MS in BPSF and BPCEs Peak area (× 106 ) BPSF Acids Alcohols Aldehydes and ketones Esters Furan(one)s Phenols Pyrazines Pyrroles Sulfurous compounds Total peak area
3.81 41.59 239.77 5.62 42.25 1.87 195.22 5.75 19.21 555.09
± ± ± ± ± ± ± ± ± ±
0.45a 3.58a 23.63a 0.59a 4.21c 0.12a 17.99a 0.44a 1.79a 52.12a
BPPTF 2.22 4.91 231.11 4.80 84.95 2.03 23.60 0.73 10.62 364.97
± ± ± ± ± ± ± ± ± ±
0.38b 0.61b 21.62ab 0.51b 8.06b 0.15a 2.20b 0.08b 1.18b 34.12b
BPPPF 0.92 3.38 117.79 2.10 11.76 0.36 6.69 0.87 4.17 148.04
± ± ± ± ± ± ± ± ± ±
0.16c 0.52b 10.93c 0.17d 1.40d 0.05b 0.69bc 0.09b 0.45c 14.23c
BPPT 3.21 6.78 202.69 3.59 101.69 0.15 6.10 0.32 10.33 334.86
± ± ± ± ± ± ± ± ± ±
0.27a 0.86b 18.40b 0.34c 8.40a 0.03c 0.50c 0.06c 1.05b 29.02b
Values are expressed as means ± standard deviations (n = 3); values in the same row followed by different letters are significantly different (P < 0.05).
above beef flavours due to their obvious differences in aroma compounds and percentages. The peak areas of different kinds of aroma compounds in BPSF and BPCEs are shown in Table 2. The peak areas of alcohols, esters, pyrazines, pyrroles, sulfurous compounds and the total peak area in BPSF were significantly higher than those in BPCEs (P < 0.05). Although the percentages of aldehydes and ketones in BPPTF, BPPPF and BPPT were about 63%, 80% and 61%, respectively, which were higher than that in BPSF, the peak area of aldehydes and ketones in BPSF was higher than that in BPCEs. The above results indicated that BPSF produced higher contents of aroma compounds, especially pyrazines and sulfurous compounds. To understand the aroma differences between BPSF and BPCEs, it was important to investigate the origins of the aroma compounds. The combination of lipid oxidation, Maillard reaction and microbial activities should be responsible for the aroma characteristics of BPSF.
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Aroma compounds from lipid oxidation Aroma compounds derived from lipid oxidation, including aliphatic aldehydes (hexanal, tetradecanal), certain ketones (3-penten-2-one, 2,3-heptanedione, 2-heptanone, 2,3octanedione, 2,6-dimethyl-3-heptanone, 2-octanone), aliphatic alcohols (2-propen-1-ol, 1-penten-3-ol, 1-hexanol, 1-octen-3-ol, 2-ethylhexanol, 1-octanol, dodecyl alcohol) and aliphatic acids (hexanoic acid, octanoic acid, tetradecanoic acid), were detected in this work. In addition, some long-chain acids and alcohols in esters were also derived from products of lipid oxidation, such as 1-hexanol in hexyl acetate, hexanoic acid in ethyl hexanoate and hexadecanoic acid in ethyl hexadecanoate. 2-Alkylfurans formed from the corresponding α,β-unsaturated aldehydes were also lipid derived.20 Most of the above aroma compounds had great relevance to the characteristic aroma since lipid oxidation products were confirmed to play an important role in the sapid and aromatic characteristics of BP.2 In this work, although a total of five acids (Table 3) were detected in four BPs, only two acids (butyric acid and tetradecanoic acid) were detected in BPSF. However, sensory evaluation indicated that BPSF possessed a stronger sour attribute than BPCEs, even though the latter contained more varieties and higher percentages of acids. The lower threshold and higher content (peak area, Table 2) of butyric acid and some acids (i.e. acetic acid) undetected in this work due to the limitation of SPME-GC-MS might be responsible for
this phenomenon. The occurrence of acetates in Table 3 confirmed the existence of acetic acid in BPSF and BPCEs. As shown in Tables 2 and 3, the peak area (content) and percentage of alcohols in BPSF were about 8.5- and 5.6, 12.3- and 3.3-, 6.1- and 3.7-fold of those in BPPTF, BPPPF and BPPT, respectively. In particular, the percentage of ethanol in BPSF was about 4.4-, 3.7- and 3.6-fold of that in BPPTF, BPPPF and BPPT, respectively. The results were consistent with the sensory evaluation result concerning alcoholic attribute (Fig. 1). In addition, 1-octen-3-ol, 2-ethylhexanol and phenethyl alcohol – aroma-active compounds in beef flavour2 and soy sauce11,14 – were detected in BPSF with much higher percentages. These compounds, especially 1-octen-3-ol, can show a strong modification to meat aroma or intensify the perception of samples associated with ‘beefy’ and ‘meaty’ aromas.2 As shown in Table 3, 28, 30, 24 and 27 aldehyde and ketone aroma compounds were detected, in which BPSF possessed the lowest percentage of aldehydes and ketones when compared with BPCEs. Individually, 2,6-dimethyl-3-heptanone (7.14%), 2-phenyl-2-butenal (4.47%), 3-methylbutanal (4.46%), acetone (3.93%) and 2-methylbutanal (3.81%) were the most abundant compounds of aldehydes and ketones in BPSF, whereas 3-methylbutanal (13.07–22.24%), benzaldehyde (3.73–9.68%), 2-methylbutanal (2.67–9.57%), acetone (3.60–9.51%) and 2butanone (3.95–9.18%) constituted the majority of aldehydes and ketones in BPCEs. Most of the above aroma compounds, such as 3-methylbutanal, 2-methylbutanal and benzaldehyde, were aroma-active compounds in beef flavour2 and soy sauce.11,14 Thus the discrepancies in percentages and compositions of aldehydes and ketones between BPSF and BPCEs would lead to their differences in aroma profiles to some extent. As shown in Table 3, only 2-methylfuran (0.27%) and 2phenylfuran (0.26%) were detected in BPSF. However, there were more varieties and a higher proportion of 2-alkylfurans, such as 2-ethylfuran (0.15–0.27%), 2-pentylfuran (0.12–0.27%) and 2phenylfuran (0.10–2.24%), detected in BPCEs. The differences in 2-alkylfuran compositions of BPSF and BPCEs might partly result in their aroma differences, such as their roast, burnt and meaty attributes.2,20 In this work, only four esters with low total peak area (5.62 × 106 ) and percentage (1.03%) were detected in BPSF. Similar results were also observed in BPCEs and previous studies.2,4 Esters, especially ethyl esters, in food could contribute a good flavour by minimizing
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Table 3. Aroma compounds and their percentages detected by SPME-GC-MS in BPSF and BPCEs Percentages of peak areas (%) Compounds
RIa
IDb
Acids Butyric acid Hexanoic acid Octanoic acid 2-Hydroxycinnamic acid Tetradecanoic acid Total (5)
1607 1823 2026 2324 2688
A,B A,B A,B A A,B
Rancid/sweaty Sour/pungent Sweaty, sour, grassy — Waxy, oily
