Monosodium glutamate equivalents and B-group vitamins in frozen mushrooms

ABSTRACT

In mushrooms the dominant taste is umami, which is created by monosodium glutamate (MSG)-like amino acids and 5’-nucleotides. In this research, the levels of 5’-nucleotides, free amino acids (liquid chromatography with mass spectrometry; LC-MS), and vitamins from the B-group (high-performance liquid chromatography; HPLC) in frozen white and brown Agaricus bisporus were determined. Before freezing, the mushrooms were blanched in water, and then in a sodium erythorbate and citric acid solution; next these were vacuum impregnated in a sodium metabisulphite and citric acid solution and blanched again in water. The varieties of mushrooms under analysis responded to the pretreatment, freezing, and frozen storage to different degrees. The equivalent concentration of umami was in the range of 0.293–1.709 g MSG/100 g dm and was higher by 26–444% in brown than in white mushrooms. After frozen storage, in the white variety, there was a higher synthesis of 5’-nucleotides and in the brown variety, of free amino acids. The main components responsible for the umami taste in both varieties were 5’-TMP, 5’-UMP, and L-glutamic acid. The white variety contained a higher level of vitamin B₂ and a lower level of vitamins B₁ and B₆.

KEYWORDS:

• Mushrooms
• Umami
• 5’-nucleotides
• Free amino acids
• Vitamins
• Freezing

Introduction

Edible mushrooms, due to their unique sensory qualities, and increasingly more often due to their high level of biological activity, are consumed all over the world. Since 1997, there has been a sharp increase in their consumption from about 1 kg to 4 kg per person per year, with the highest quantities of mushrooms being consumed in Asian countries, mainly China.^[1] In the countries of the Far East, edible mushrooms are classified as functional foods as they contain many biologically active compounds, including vitamins from the B-group and vitamin D, minerals, phenolic compounds, polyketides, terpens, steroids, and dietary fibre, including beta-glucans.^[2–5]

Many of the B vitamins, especially thiamin, are sensitive to degradation during processing and therefore they can be considered as an indicator of food quality.^[2] Thiamin is more sensitive to being heated up than riboflavin, and moreover, it is barely stable in an alkaline environment, whereas riboflavin is stable with respect to both oxidation and heat, but sensitive to light, and vitamin B₆ is resistant to heat, acid, and alkaline, but sensitive to light in neutral and alkaline solutions. Moreover, the thermal degradation of vitamin B₆ increases as the pH rises.^[6] With respect to the above and considering the alkaline character of mushrooms, it is important to set up an acidic environment during their preliminary treatment.

The fruiting bodies of edible mushrooms are characterized by a unique taste and flavour. A mushroom’s taste is dependent on volatile and non-volatile compounds, and among the non-volatile compounds we can include 5’-nucleotides, free amino acids, and soluble carbohydrates.^[7] In mushrooms, the dominant taste is the so-called umami taste created by sodium salts of glutamic (Glu) and aspartic (Asp) acids (monosodium glutamate (MSG)-like amino acids) and 5’-nucleotides such as 5’-guanosine monophosphate (5’-GMP), 5’-inosine monophosphate (5’-IMP), and 5’-xanthosine monophosphate (5’-XMP).^[8,9] Nucleotides (IMP and GMP) cannot activate the umami taste receptors on their own, but they can intensify the umami sensation by several times and this is caused by glutamate; however, in the case of 5’-nucleotides and glutamate, a synergistic effect is observed.^[9] To characterize the umami-like taste of mushrooms, we may use the so-called equivalent umami concentration (EUC), which takes into account the participation of the particular compounds that create the umami taste in mushrooms.^[7]

Over the last few years, the popularity of crimini (brown A. bisporus) mushrooms has increased significantly. Crimini mushrooms, like button mushrooms, are characterized by low durability. In Poland, as well as in other countries, there is a commonly held belief that crimini mushrooms are more aromatic and tasty, and that they are characterized by a higher nutritional value than traditional button mushrooms. The scientific literature provides much information about the taste of fresh and processed mushrooms, but there is practically no information about frozen mushrooms, including crimini. ^[7–13]

With respect to the above, the aim of the research was to compare the levels of MSG equivalents (5’-nucleotides, free amino acids) and selected vitamins from the B-group (B₁, B₂, and B₆) in frozen white and brown Agaricus bisporus mushrooms. Preserved mushrooms were stored for 8 months. The MSG equivalents were analysed using liquid chromatography with mass spectrometry (LC-MS), whereas the vitamins of the B-group were analysed using high-performance liquid chromatography (HPLC). The results of the instrumental evaluation were completed with a sensory analysis of the taste using a five-point method.

Materials and methods

Materials

The materials examined consisted of fresh and frozen Agaricus bisporus (Lange) Sing. mushrooms in two varieties: (i) with caps of white colour (button), and the intermediate hybrid SP strain 251; (ii) with caps of brown colour (crimini) and strain K102. Before freezing, the mushrooms were subject to diversified preliminary treatments. The frozen mushrooms were evaluated immediately after freezing and after 8 months of storage at –25°C. Fresh mushrooms were obtained from a grower in Radostowice, Poland. The mushrooms were frozen 2 h after harvest. The fruiting bodies of both varieties had diameters of 3–5 cm, and the caps were joined to the stems by a membrane. The freezing process comprised the following stages: selection, preliminary treatment, freezing, and frozen storage. The preliminary processing and freezing processes were conducted under laboratory conditions, enabling the precise control of parameters at every stage of the technological treatment. The quantities involved were consistent with a small-scale investigation, 20 kg of mushrooms being used to obtain each type of examined product.

Preliminary treatment consisted of: cleaning and sorting; rinsing under running tap water; and slicing with a knife into approximately 10-mm strips. One part of the sliced mushrooms was frozen (product code Not blanched), while the other part was subjected to three kinds of pretreatment:

1. blanching in water (product code BW),

2. vacuum impregnation in a sodium metabisulphite (0.2 g/100 g) and citric acid (0.5 g/100 g) solution and then blanching in water (product code VAC+BW),

3. blanching in a sodium erythorbate (0.3 g/100 g) and citric acid (0.3 g/100 g) water solution (product code B ERYT).

Blanching was carried out for 180 s at 96–98°C in stainless steel vessels with a volume of 10 litres, the proportion of mushrooms to blanching medium being 1:5 (w/w). After blanching, the material was cooled in water and then drained on sieves for about 30 min. Vacuum impregnation was performed in a Heidolph (Germany) LABOROTA 4000 vacuum evaporator at room temperature (20–22°C) for 15 min at a pressure of 0.025 MPa. Fresh mushrooms and those after pretreatment were frozen in a single layer (thickness about 3 cm) using the air-blast method in a Feutron 3626–51 blast freezer at –40°C for 120 min in order to achieve a temperature of –25°C at the thermal centre of the sample. After freezing, the mushrooms were placed in 0.5 litre polyethylene containers. The products were stored for 8 months at –25°C.

Methods

Fresh and frozen mushrooms (directly after freezing and after 8 months of frozen storage) were subjected to analysis of the dry matter, 5’-nucleotides (5’-AMP, 5’-CMP, 5’-UMP, 5’-GMP, 5’-IMP, 5’-TMP), L-aspartic acid, and L-glutamic acid, vitamins: B₁, B₂, and B₆ (pirydoxine, pirydoxal, pirydoxamine). The dry matter content was determined using the AOAC^[14] method by drying at 105ºC to a constant weight.

5’-nucleotides and free amino acids

The quality and quantity analysis of 5’-nucleotides and free amino acids was carried out using an LC-MS method with modifications.^[15] The freeze-dried mushrooms (500 mg) were subjected to extraction with water of HPLC grade with the use of sonification in an ultrasound InterSonic IS-14 bath at an ultrasound frequency rate of 35 kHz at 20°C for 15 min. The prepared extracts were centrifuged (5000 rpm/15 min) and filtered through 0.22-μm PTFE filters (polypropylene housing (Sigma-Aldrich)) and analysed using an LC-MS chromatograph. The column was thermostated at a temperature of 30°C (5’-nucleotides) or 60°C (free amino acids). In the case of the 5’-nucleotides, isocratic elution was carried out using a 0.1% formic acid solution for 17 min at a flow rate of 0.40 ml/min. In the case of the free amino acids, 0.1% formic acid and methanol were used as the mobile phase in the gradient elution (t = 0 min. fa/met 95/5; t = 10 min. fa/met 90/10) with a flow rate of 0.3 cm³/min.

The Shimadzu (Tokyo, Japan) LC-MS system was used, equipped with a dual LC-30AD pump, Nexera-X2 (SPDM-30A), LC-MS 2020 mass detector with ESI detection system, and a CBM-20Alite controller unit. In the case of the 5’-nucleotides, simultaneous positive and negative ionization in the SCAN mode – full-scan mass spectrum (m/z 300–400) – and SIM mode (m/z for standards) was carried out. In the case of free amino acids, positive ionization in the SIM mode (m/z for standards) was carried out. The parameters of the analysis were as follows: core interface temperature 350°C, desolvation line temperature 300°C, nebulizing gas (nitrogen) flow 1.5 l/min, drying gas (nitrogen) flow 10 l/min, and the temperature of the heat block was set to 350°C. For the separation of nucleotides and free amino acids, a Kinetex XB-C18 column was used (Phenomenex, 1.7 µm, 150 × 2.1 mm). Calibration curves were prepared using appropriate standards in the concentration range from 5 to 500 µg/ml.

Identification of 5’-nucleotides and free amino acids was performed based on their retention time and MS spectra in accordance with the standards (5’-AMP, 5’-CMP, 5’-UMP, 5’-GMP, 5’-IMP, 5’-TMP, L-aspartic acid, L-glutamic acid). Quantification was performed according to the calibration curves prepared for the standards in the range of concentration of 5–500 µg/ml. The standards of 5’-nucleotides and free amino acids were bought from the Sigma–Aldrich company, and their cleanness was: adenosine 5′-monophosphate monohydrate > 99%, cytidine 5′-monophosphate disodium salt > 99%, guanosine 5′-monophosphate disodium salt hydrate > 99%, inosine 5′-monophosphate > 98%, uridine 5′-monophosphate >98–100%, thymidine 5′-monophosphate disodium salt hydrate > 99%, L-aspartic acid > 98%, and L-glutamic acid > 99%.

The EUC (g MSG per 100 g of dry matter) was calculated based on the following equation: Y = Ʃa_ib_i + 1218 (Ʃa_ib_i)(Ʃa_jb_j), where Y is the EUC of the mixture in terms of g MSG per 100 g dm; a_i is the concentration of each umami amino acid (L-aspartic acid or L-glutamic acid); a_j is the concentration (g per 100 g dm) of each umami 5’-nucleotide (5’-IMP, 5’-GMP, 5’-AMP); b_i is the relative umami concentration (RUC) for each umami amino acid to MSG (Glu = 1, Asp = 0.077); b_j is the RUC for each umami 5’-nucleotide (5’-IMP = 1, 5’-GMP = 2.3, 5’-AMP = 0.18); and 1218 is a synergistic constant based on the concentration of g per 100 g of dry matter.^[7]

B-group vitamins

Vitamin B₁ (thiamine) and B₂ (riboflavin) contents were determined using HPLC.^[16,17] Thiamine and riboflavin were determined simultaneously using a Merck liquid chromatograph with a fluorescence detector. The analysis was carried out on an Onyx Monolithic C18 column (100 x 4.6 mm) with pre-column (both Phenomenex, Torrance, USA), and was conducted at an excitation and emission wavelength of 360/503 nm. Water and acetonitrile were used as a mobile phase in the gradient elution (t = 0 w/ac 88/12; t = 12 w/ac 0/100) with a flow rate of 0.9 cm³/min.

Vitamin B₆ content was determined using the HPLC method.^[18] The analysis was carried out on a Merck liquid chromatograph fitted with a fluorescence detector on a Phenosphere-Next C18 (150 × 4.60 mm) monolithic column with pre-column (both Phenomenex). Isocratic elution with a flow rate of 0.9 cm³/min was performed using a solution of sulphuric acid (c = 0.015 mol/l) and trichloroacetic acid (TCA) of ≥99% purity (c = 0.005 mol/l). Measurements were performed at excitation/emission wavelengths of 290/390 nm.

Sensory evaluation

The sensory quality of mushroom samples, previously defrosted at 2–4°C for 12 h, was evaluated directly after freezing and after 8 months of frozen storage using a five-point scale method. Mushroom quality was scored on a 5-1 scale (5 = excellent; 4 = very good; 3 = good; 2 = bad; 1= very bad) by a panel of five judges fulfilling the requirements for sensory sensitivity according to Polish standards.^[19] The lowest acceptable score for any characteristic assessed by the sensory panel was set at 3.0. The samples were evaluated in terms of taste.

Statistical analysis

All samples were analysed in three independent replications (n = 3). Results were analysed statistically using a single-factor analysis of variance (ANOVA) on the basis of Duncan’s range test (p < 0.05). Calculations were performed using Statistica 10.0 Pl (Stat-Soft).

Results and discussion

Dry matter

The results obtained for fresh and frozen mushrooms showed that 100 g of fruiting body contained 7.70–10.29 g of dry matter, and it should be specified that considerably higher amounts (more by 15–22%) were determined in the case of button than crimini mushrooms (Table 1). The highest amount of dry matter was determined in blanched frozen mushrooms (products BW, B ERYT.). According to Reis et al.^[20] fresh Agaricus bisporus contained an amount of dry matter (8.73 g), which was typical for this species. In comparison to the previous research of the author of the manuscript^[2,21] and the research conducted by Li et al.,^[22] the determined level of dry matter in fresh button mushrooms was higher by 1.33–1.63 g. A similar dependency was also indicated with respect to frozen materials^[2] for which the differences were as high as 4.64–5.58 g. The differences that were determined in the contents of dry matter may have resulted above all from the application of different strains of Agaricus bisporus and different parameters of the preliminary treatment.

Table 1. Dry matter (in 100 g fm) and vitamins (in 100 g dm) in fresh and frozen mushrooms.

		White – Bottom			Brown – Crimini
	Pretreatment	Fresh	0 months	8 months	Fresh	0 months	8 months
Dry matter [g]	Not blanched	8.92 ± 0.05f	8.95 ± 0.06f	8.96 ± 0.06f	7.70 ± 0.04a	7.79 ± 0.04b	7.79 ± 0.02b
	BW		10.27 ± 0.03i	10.29 ± 0.05i		8.67 ± 0.04e	8.64 ± 0.05e
	VAC+BW		9.80 ± 0.02g	9.79 ± 0.11g		8.04 ± 0.06c	8.03 ± 0.02c
	B ERYT		10.18 ± 0.03h	10.19 ± 0.05h		8.43 ± 0.07d	8.42 ± 0.02d
Vitamin B1 [mg]	Not blanched	0.63 ± 0.01j	0.53 ± 0.01hi	0.46 ± 0.10efg	0.55 ± 0.01i	0.49 ± 0.01gh	0.48 ± 0.03fg
	BW		0.41 ± 0.01cde	0.36 ± 0.03bc		0.45 ± 0.01efg	0.43 ± 0.03def
	VAC+BW		0.37 ± 0.00bc	0.27 ± 0.01a		0.38 ± 0.01bcd	0.37 ± 0.02bcd
	B ERYT		0.38 ± 0.01bcd	0.34 ± 0.01b		0.37 ± 0.01bcd	0.34 ± 0.07b
Vitamin B2 [mg]	Not blanched	6.11 ± 0.26n	5.46 ± 0.09m	5.26 ± 0.21lm	4.62 ± 0.08j	4.19 ± 0.01fg	3.34 ± 0.11b
	BW		5.10 ± 0.06kl	4.93 ± 0.03jk		4.30 ± 0.02g	3.58 ± 0.03c
	VAC+BW		4.02 ± 0.17ef	3.88 ± 0.18de		3.72 ± 0.23cd	2.71 ± 0.03a
	B ERYT		4.79 ± 0.09ij	4.55 ± 0.15h		3.31 ± 0.01b	2.63 ± 0.07a
Pyridoxamine [µg]	Not blanched	313 ± 2l	173 ± 4g	106 ± 1a	247 ± 12ab	166 ± 12fg	144 ± 9cd
	BW		169 ± 6g	152 ± 2de		138 ± 1c	123 ± 2b
	VAC+BW		260 ± 3k	220 ± 2i		173 ± 2g	146 ± 3cd
	B ERYT		157 ± 2ef	150 ± 6de		215 ± 13i	190 ± 1h
Pyridoxal [µg]	Not blanched	11 ± 2ab	13 ± 1cd	Nd	9 ± 1ab	8 ± 1a	34 ± 2h
	BW		36 ± 3h	11 ± 1bc		20 ± 2f	75 ± 1l
	VAC+BW		36 ± 3h	50 ± 1j		14 ± 1d	62 ± 2k
	B ERYT		23 ± 1g	11 ± 1ab		17 ± 1e	40 ± 3i
Pyridoxine [µg]	Not blanched	32 ± 3b	nd	nd	19 ± 1a	40 ± 2c	nd
	BW		nd	nd		61 ± 5d	nd
	VAC+BW		nd	nd		nd	nd
	B ERYT		nd	nd		33 ± 2b	nd
Sum of vitamin B6 [µg]	Not blanched	356 ± 4j	186 ± 4c	106 ± 1a	275 ± 13h	214 ± 14ef	178 ± 10c
	BW		205 ± 3de	163 ± 2b		219 ± 4f	198 ± 2d
	VAC+BW		296 ± 2i	270 ± 3h		187 ± 3c	207 ± 3de
	B ERYT		180 ± 2c	161 ± 6b		265 ± 12h	230 ± 4g

BW – blanched in water; VAC+BW – vacuum impregnated by metabisulphite and citric acid solution, next blanched in water; B ERYT – blanched in sodium erythorbate and citric acid solution; fm – fresh matter; dm – dry matter; nd – not detected; means with different small letters represent significant differences at p < 0.05.

5’-nucleotides

The fruiting bodies of edible mushrooms are characterized by a unique taste and aroma, and it should be specified that the level of compounds responsible for these features depends on, among other things, the species of the mushroom, the part, the degree of maturity of the fruiting body, and the method of treatment.^[7,11,22-25] In the scientific literature, one may find considerable divergence with respect to the level of 5’-nucleotides. For instance, Sommer et al.^[15] state that in fresh Agaricus bisporus there is 75 mg/100 g dm 5’-AMP, whereas according to Li et al.^[22] the quantity of this compound in Agaricus blazei is almost twice as high. On the other hand, Yang et al.^[25] proved that in Lentinula edodes mushrooms there is no 5’-AMP at all, whereas in Pleurotus ostreatus it is at the level of 437 mg/100 g dm. Fresh button (white variety) and crimini (brown variety) mushrooms did not differ to a significant degree in the sum of 5’-nucleotides. Considerable differences were, however, proved in general with respect to the levels of 5’-TMP, 5’-CMP, and 5’-IMP. In button mushrooms, 5’-TMP and 5’-UMP prevailed, whereas in crimini, it was 5’-TMP (Table 2). In both varieties the lowest quantity was that of 5’-AMP. Li et al.^[22], in fresh Agaricus blazei, determined a lower level of about 2 mg sum of 5’-nucleotides with the highest content of 5’-CMP and the lowest content of 5’-UMP. In addition, the authors stated that among the species of mushrooms under analysis, the highest level of 5’-nucleotides is contained in the Coprinus comatus and the lowest in Pleurotus erynii mushrooms. On the other hand, Tseng and Mau^[26], in fresh A. bisporus, determined a level of nucleotides several times higher than in the research under discussion, as their sum was 419 mg/100 g dm, where 5’-CMP prevailed.

Table 2. 5’-nucleotides in fresh and frozen mushrooms, mg/100 g dm.

		White – Bottom			Brown – Crimini
	Pretreatment	Fresh	0 months	8 months	Fresh	0 months	8 months
5’-TMP	Not blanched	3.85 ± 0.27e	2.07 ± 0.19d	0.72 ± 0.01a	7.51 ± 0.63g	6.99 ± 0.17f	0.73 ± 0.02a
	BW		1.11 ± 0.07bc	1.18 ± 0.05c		1.08 ± 0.04abc	0.94 ± 0.07abc
	VAC+BWW		0.72 ± 0.02a	0.76 ± 0.01ab		0.91 ± 0.09abc	0.87 ± 0.05abc
	B ERYT		0.95 ± 0.05abc	1.00 ± 0.06abc		0.98 ± 0.08abc	0.95 ± 0.05abc
5’-CMP	Not blanched	2.86 ± 0.14d	3.89 ± 0.18e	3.81 ± 0.22e	1.08 ± 0.05b	1.91 ± 0.11c	0.24 ± 0.04a
	BW		8.23 ± 0.74g	12.64 ± 0.74j		6.53 ± 0.29f	7.06 ± 0.62f
	VAC+BW		1.62 ± 0.15bc	2.25 ± 0.15cd		0.17 ± 0.06a	4.13 ± 0.30e
	B ERYT		9.73 ± 0.88h	10.96 ± 0.22i		6.95 ± 0.45f	7.14 ± 0.67f
5’-UMP	Not blanched	3.63 ± 0.34ab	7.72 ± 0.69cd	5.34 ± 0.42abc	2.81 ± 0.20a	2.81 ± 0.24a	3.24 ± 0.20a
	BW		53.35 ± 3.13k	47.23 ± 2.27j		14.67 ± 1.14g	5.90 ± 0.27bc
	VAC+BW		12.56 ± 0.90fg	11.07 ± 0.84ef		4.74 ± 0.07ab	7.87 ± 0.38cd
	B ERYT		40.26 ± 3.53i	23.56 ± 1.74h		12.62 ± 1.04fg	9.25 ± 0.86de
5’-AMP	Not blanched	0.65 ± 0.04a	2.10 ± 0.08c	4.65 ± 0.33e	0.79 ± 0.03ab	3.78 ± 0.21d	0.74 ± 0.04ab
	BW		1.33 ± 0.14abc	1.15 ± 0.03ab		1.63 ± 0.14bc	0.57 ± 0.06a
	VAC+BW		0.98 ± 0.08ab	0.58 ± 0.04a		20.24 ± 2.02g	6.96 ± 0.49f
	B ERYT		1.03 ± 0.10ab	0.97 ± 0.13ab		1.42 ± 0.08abc	1.53 ± 0.07abc
5’-IMP	Not blanched	2.80 ± 0.21g	2.46 ± 0.04bcdef	2.49 ± 0.02def	2.56 ± 0.08f	2.47 ± 0.04cdef	2.46 ± 0.01abcdef
	BW		2.26 ± 0.03abc	2.25 ± 0.01ab		2.32 ± 0.08abcde	2.42 ± 0.06abcdef
	VAC+BW		2.52 ± 0.07ef	2.60 ± 0.04f		4.64 ± 0.25i	3.12 ± 0.26h
	B ERYT		2.28 ± 0.07abc	2.25 ± 0.01ab		2.29 ± 0.05abcd	2.29 ± 0.03abcd
5’-GMP	Not blanched	1.19 ± 0.07a	2.00 ± 0.19d	1.13 ± 0.10a	1.29 ± 0.11ab	1.29 ± 0.10ab	1.11 ± 0.03a
	BW		1.46 ± 0.15bc	1.58 ± 0.11c		1.13 ± 0.02a	1.14 ± 0.02a
	VAC+BW		1.06 ± 0.01a	1.10 ± 0.01a		5.82 ± 0.21f	3.99 ± 0.35e
	B ERYT		1.14 ± 0.08a	1.58 ± 0.09c		1.13 ± 0.01a	1.13 ± 0.02a
Total 5’-nucleotides	Not blanched	14.98 ± 0.35b	20.24 ± 0.74de	18.14 ± 0.90bcd	16.04 ± 1.52bc	19.25 ± 1.07cde	8.51 ± 0.22a
	BW		67.74 ± 3.50j	66j.04 ± 2.95j		27.37 ± 0.71f	18.03 ± 0.88bcd
	VAC+BW		19.46 ± 0.97cde	18.35 ± 0.80bcd		36.53 ± 1.98g	26.94 ± 0.41f
	B ERYT		55.39 ± 4.72i	40.31 ± 1.57h		25.39 ± 1.99f	22.29 ± 1.54e

BW – blanched in water; VAC+BW – vacuum impregnated by metabisulphite and citric acid solution, next blanched in water; B ERYT – blanched in sodium erythorbate and citric acid solution; dm – dry matter; means with different small letters represent significant differences at p < 0.05.

As a result of freezing and frozen storage in general, a considerable increase in the sum of 5’-nucleotides (by 21–340% in button mushrooms and by 13–68% in crimini) was found, compared with the raw material. The main reason for this phenomenon was an increase of 1–6 times in the quantity of 5’-CMP and of 1–12 times in the quantity of 5’-UMP. Considering the amounts of the other 5’-nucleotides at the end of the storage, it was determined that in both varieties the quantity of 5’-TMP decreased significantly by 69–90% and, in general, the quantity of 5’-IMP decreased by 4–20%. In addition, changes in the amounts of 5’-GMP and 5’-AMP within the ranges, respectively, from –14 to +351% and from –11 to +781% were observed. At the end of the frozen storage, with the exception of the VAC+BW product, unlike in the case of the raw material, the button mushrooms were characterized by a significantly higher (by 81–266%) sum of 5’-nucleotides, compared with the crimini, including in general higher levels of 5’-CMP, 5’-UMP, and 5’-GMP. The kind of preliminary treatment applied before freezing had, in general, a considerable effect on the levels of the 5’-nucleotides, and it should be specified that they also depended on the variety of mushrooms. In the case of button mushrooms, the highest sum of 5’-nucleotides was found in mushrooms that had been blanched in water, whereas in the case of crimini this was the highest with mushrooms that had been vacuum impregnated and then blanched. The changes in the quantities of individual 5’-nucleotides might have been caused by their transformations from one form of the 5’-nucleotide into another due to pretreatment, freezing, and frozen storage. According to Dudzińska and Hłyńczak^[27] and Nagodawithana,^[28] 5’-AMP, 5’-IMP, and 5’-GMP participate in the so-called purine nucleotides cycle, as a result of which with the participation of tissue enzymes and ATP they may transform from one into another, e.g. deamination of 5’-AMP to 5’-IMP or re-amination of 5’-IMP to 5’-AMP.

Free amino acids

Mushrooms also contain significant quantities of free amino acids, which may comprise from 0.2 up to 7.2 g/100 g of the dry matter.^[29,30] The dominant free amino acids are glutamic acid, ornithine, and alanine.^[31] Free amino acids have an important role in the creation of the taste of mushrooms, mainly including glutamic and aspartic acids, which together with 5’-nucleotides create the so-called umami taste.^[32] Moreover, α-amino acids are precursors of many compounds that contain nitrogen, such as haeme, glutathione, hormones, and nucleotides.^[33] The quantities of free amino acids are affected by a number of factors including, among other things, the age of fruiting bodies and the time of their storage. According to Mau et al.,^[23] in the fruiting bodies of V. volvacea with open caps, there are almost twice as many free amino acids than in mushrooms with closed caps. Tseng and Mau^[26] state that the storage of A. bisporus at a temperature of 12°C for 12 days causes the sum of free amino acids in the fruiting bodies to double. Fresh mushrooms of both varieties differed in the levels of free amino acids, and it should be specified that a higher quantity (by 43–44%) was found in crimini (brown variety) than in button (white variety) mushrooms (Table 3). In both varieties, L-glutamic acid prevailed. As a result of the process of technological freezing and frozen storage, in general, low and insignificant changes in the quantity of free amino acids in button mushrooms were observed. In the case of crimini, in general, there was a significant increase in the amount of L-aspartic acid (by 10–49%) and L-glutamic acid (by 3–16%), compared with the raw mushrooms. As with the raw mushrooms, a higher (by 20–127%) quantity of the compounds under discussion was observed in crimini than in button mushrooms. In both varieties, L-glutamic acid was the most prevalent, with a quantity higher (by 38–72%) than that of L-aspartic acid. The preliminary treatment applied before freezing had an effect on the quantity of free amino acids in the mushrooms. Regardless of the variety of Agaricus bisporus mushrooms, the lowest level of free amino acids was found in the products from unblanched fruiting bodies, and it should be specified that higher disproportions between products were determined in crimini than in button mushrooms.

Table 3. L-aspartic acid and L-glutamic acid in fresh and frozen mushrooms, mg/100 g dm.

		White – Bottom			Brown – Crimini
	Pretreatment	Fresh	0 months	8 months	Fresh	0 months	8 months
L-aspartic acid	Not blanched	19.2 ± 1.7abc	16.2 ± 1.7a	16.7 ± 1.3ab	27.5 ± 2.3e	24.4 ± 1.4d	23.1 ± 2.1d
	BW		28.4 ± 1.8e	20.0 ± 1.6c		34.1 ± 1.3f	30.3 ± 2.1e
	VAC+BWW		18.3 ± 0.9abcc	18.1 ± 0.6abc		39.5 ± 1.8g	41.0 ± 3.1g
	B ERYT		19.9 ± 1.3bc	19.4 ± 1.0abc		34.0 ± 1.0f	35.4 ± 2.4f
L-glutamic acid	Not blanched	38.7 ± 3.1ab	35.1 ± 2.4a	34.8 ± 2.0a	55.7 ± 1.2de	57.6 ± 4.0ef	52.9 ± 2.5d
	BW		39.3 ± 2.2ab	41.4 ± 0.8b		62.7 ± 0.7gh	64.5 ± 4.6h
	VAC+BW		41.9 ± 2.1b	47.7 ± 0.6c		59.3 ± 1.3efg	57.2 ± 2.3def
	B ERYT		39.2 ± 1.6ab	42.9 ± 0.5b		59.5 ± 4.0efg	61.7 ± 4.6fgh
Sum of amino acids	Not blanched	58.0 ± 4.8ab	51.3 ± 4.0a	51.5 ± 3.3a	83.2 ± 2.5f	82.0 ± 5.3ef	75.9 ± 4.5e
	BW		67.6 ± 4.0d	61.5 ± 2.3bcd		96.8 ± 1.1g	94.8 ± 6.5g
	VAC+BW		60.3 ± 3.0bc	65.8 ± 1.1cd		98.8 ± 3.1g	98.2 ± 3.2g
	B ERYT		59.1 ± 2.6bcd	62.3 ± 1.3bcd		93.5 ± 5.0g	97.1 ± 6.9g

BW – blanched in water; VAC+BW – vacuum impregnated by metabisulphite and citric acid solution, next blanched in water; B ERYT – blanched in sodium erythorbate and citric acid solution; dm – dry matter; means with different small letters represent significant differences at p < 0.05.

Non-volatile compounds

5’-nucletides and free amino acids are responsible for the umami or palatable taste,^[34] and among these 5’-nucleotides there are: 5’-AMP, 5’-IMP, 5’-GMP, and 5’-XMP.^[32] The quoted authors also state that L-aspartic and L-glutamic acids as well as 5’-nucleotides present in mushrooms may cause a synergistic effect. Fresh mushrooms of both varieties did not differ in the levels of flavour 5’-nucleotides (Table 4), although it should be noted that compared with Agaricus blazei, Agrocybe cylindracea, Pleurotus cystidiosus, or Pleurotus eryngii analysed by Li et al.,^[22] they were a few times more aromatic. As a result of freezing and frozen storage, in general, low and insignificant changes in the level of flavour 5’-nucleotides were observed, when compared with the raw mushrooms. At the end of storage, frozen button mushrooms were characterized by a slightly higher level of the flavour 5’-nucleotides than in the crimini mushrooms, although an exception was noted for vacuum-impregnated frozen mushrooms (VAC+BW), where an inverse relationship was observed. The preliminary treatment applied before freezing had an effect on the quantity of flavour 5’-nucleotides, but it also depended on the variety of Agaricus bisporus. In the case of button mushrooms, the highest level of flavour 5’-nucleotides was found in those blanched in water (BW) or in sodium erythorbate (B ERYT) frozen products, whereas in the case of crimini the highest level was found in those that had been vacuum impregnated (VAC+BW). According to the definition provided by Yang et al.,^[25] the fresh and frozen Agaricus bisporus may be qualified as in the low range (< 100 mg/100 g dm) with respect to the content of flavour 5’-nucleotides.

Table 4. Flavour nucleotides, equivalent umami concentration (EUC), and taste by five-point sensory analysis in fresh and frozen mushrooms.

		White – Bottom			Brown – Crimini
	Pretreatment	Fresh	0 months	8 months	Fresh	0 months	8 months
Flavour 5’-nucleotides [mg/100 g dm]	Not blanched	3.99 ± 0.25d	4.46 ± 0.10e	3.63 ± 0.05abcd	3.85 ± 0.04cd	3.76 ± 0.09abcd	3.57 ± 0.03abc
	BW		3.73 ± 0.18abcd	3.83 ± 0.12bcd		3.45 ± 0.07ab	3.56 ± 0.07abc
	VAC+BW		3.58 ± 0.07abc	3.69 ± 0.04abcd		10.46 ± 0.39g	7.11 ± 0.61f
	B ERYT		3.42 ± 0.15a	3.83 ± 0.09bcd		3.42 ± 0.05a	3.42 ± 0.05a
EUC [g MSG/100 g dm)	Not blanched	0.317 ± 0.012ab	0.365 ± 0.013cd 0.01bc5	0.297 ± 0.013ab Cd	0.457 ± 0.006e	0.502 ± 0.040f Ef	0.397 ± 0.019d Ef
	BW		0.338 ± 0.015bc a	0.362 ± 0.007cd cd		0.480 ± 0.010ef e	0.486 ± 0.031ef
	VAC+BWW		0.314 ± 0.013ab 0.026	0.362 ± 0.006cd		1.709 ± 0.035h	1.057 ± 0.050g
	B ERYT		0.293 ± 0.026a	0.372 ± 0.013cd		0.451 ± 0.024e	0.470 ± 0.029ef
Taste [points]	Not blanched	5.0i	2.0 ± 0.0a	2.0 ± 0.0a	5.0i	3.1 ± 0.4b	2.0 ± 0.0a
	BW		4.6 ± 0.1h	3.8 ± 0.1c		4.7 ± 0.2h	4.2 ± 0.1efg
	VAC+BW		3.0 ± 0.1b	3.9 ± 0.2cd		4.1 ± 0.1def	3.8 ± 0.1c
	B ERYT		4.3 ± 0.1fg	4.2 ± 0.1efg		4.4 ± 0.2g	4.0 ± 0.3cde

BW – blanched in water; VAC+BW – vacuum impregnated by metabisulphite and citric acid solution. next blanched in water; B ERYT – blanched in sodium erythorbate and citric acid solution; dm – dry matter; MSG – monosodium glutamate; flavour 5’-nucleotides – sum of GMP + IMP; means with different small letters represent significant differences at p < 0.05.

EUC index –MSG equivalents

According to Tsai et al.,^[35] the EUC index, which is an equivalent of MSG, may be divided into three levels: >2 g MSG/100 g dm (1 level), 0.5−2.0 g MSG/100 g dm (2 level), <0.5 g MSG/100 g dm (3 level), although it should be noted that its value is affected to a considerable degree by the method of extraction of 5’-nucleotides and free amino acids from the fruiting bodies. According to Dermiki et al.,^[8] along with an increase in the temperature of extraction from 22 to 70°C and an extension of the time of extraction from 30 to 360 min, the EUC index assumes values from 0.27 g MSG/100 g dm (22°C/30 min) to 22.6 g MSG/100 g dm (70°C/360 min). Moreover, according to Wu et al.,^[24] the value of the EUC index is considerably affected by the method of drying as the highest level of EUC was observed in the fruiting bodies of Hypsizygus marmoreus, which had been subjected to freeze-drying, and the lowest in those subjected to vacuum drying and microwave-vacuum drying. In the manuscript, the value of EUC for fresh Agaricus bisporus, depending on the variety, was within level 3 as it was 0.32–0.46 g/100 g dm (Table 4). A higher level on the EUC index (by 44%) was determined in crimini (brown variety) than in button (white variety) mushrooms. The values of EUC obtained in the research were close to the level provided by Dermiki et al.^[8] for shiitake mushrooms subjected to extraction at a temperature of 22°C for 60 min (0.37 g MSG/100 g dm). According to Cho et al.,^[7] in Tricholoma matsutake mushrooms, the EUC index is at a significantly lower level, i.e. 0.09–0.20 g MSG/100 g dm in pileus and 0.01–0.09 g MSG/100 g dm in stipe. As a result of the preliminary treatment and freezing, in general, insignificant changes in the EUC values in button mushrooms were observed as they ranged from –9 to +13%, compared with the raw mushrooms. In the case of crimini, the changes observed ranged from –2 to +272% and they were considerable in unblanched and vacuum-impregnated frozen fruiting bodies. As a result of the frozen storage, it was found that, except for unblanched frozen fruiting bodies, the value of the EUC index increased by 6–28% in button mushrooms, compared with un-stored products. As for crimini mushrooms, considerable changes were found only in the product from unblanched and vacuum-impregnated Agaricus bisporus where the EUC index decreased by 20–38%. The preliminary treatment applied before freezing had an effect on the level of the EUC index, and it should be noted that more considerable differences between the products were determined in crimini than in button mushrooms. In the case of crimini, the best preliminary treatment in terms of a high level on the EUC index was vacuum impregnation. Regardless of the stage of the evaluation, the variety of Agaricus bisporus had a considerable effect on the value of the EUC index. A higher level on the EUC index (by 26–444%) was determined in frozen crimini than in button mushrooms, which was also confirmed in the sensory evaluation. The frozen mushrooms were characterized by considerably lower values on the EUC index than the fresh and dried Hypsizygus marmoreus analysed by Wu et al.^[24] and several times higher than the canned A. bisporus (0.04 g MSG/100 g dm) analysed by Chiang et al.^[11] The phenomenon may be due to the fact that a different species of mushrooms or a different method of preserving was used.

Directly after freezing, except for the unblanched button mushrooms, the mushrooms were characterized as having quite a good taste (Table 4). As a result of frozen storage, a deterioration in the taste of mushrooms by 0.1–1.1 points was observed, and it should be specified that regardless of the variety, the products from the unblanched mushrooms were characterized by an unacceptable taste. In the case of button mushrooms, the taste of those frozen fruiting bodies blanched in sodium erythorbate was estimated to be the best, whereas in the case of crimini the best was the taste of the mushrooms blanched in water. Similar conclusions were drawn by Jaworska et al.^[36] who, after 8 months of frozen storage, considered as unacceptable the taste of unblanched Agaricus bisporus and the taste of the product blanched in water as the best. The variety of Agaricus bisporus had an effect on the taste of frozen mushrooms as the frozen crimini was, in general, characterized by a slightly better taste but not always significantly more than frozen button mushrooms.

Vitamins

Edible mushrooms, due to the presence of many biologically active ingredients, are qualified as functional foods. The high content of vitamins from the B-group and vitamin D is not to be underestimated.^{[2,5,30,37–39]} According to Furlani and Godoy,^[38] the fresh fruiting bodies of button (white variety) and crimini (brown variety) mushrooms differ in the amounts of vitamins B₁ and B₂, and it should be specified that a slightly higher level exists in the crimini mushrooms than in the button. In the manuscript, unlike what was stated by Furlani and Godoy,^[38] fresh and frozen button mushrooms contained considerably more vitamin B₂ (8–73%), whereas in general they did not differ in the levels of vitamin B₁, in comparison with crimini mushrooms (Table 1). Moreover, button mushrooms contained 29% more of the sum of vitamin B₆ than crimini. In the mushrooms of both varieties, the presence of all three determined forms of vitamin B₆ was found, and it should be specified that the prevailing one was pyridoxamine (78–88% of the sum of vitamin B₆), whereas the lowest quantity was that of pyridoxal (3% of the sum of vitamin B₆). As a result of the freezing, a considerable decrease in the quantity of vitamin B₁ (by 10–41%) and B₂ (by 7–34%) was found as well as, in general, a considerable decrease in the sum of vitamin B₆ (by 4–49%). Frozen storage only affected the content of vitamin B₂ to a considerable degree in crimini mushrooms (a decrease of 23–43%) and in general of vitamin B₆ in both varieties. At the end of the frozen storage, the sum of vitamin B₆ decreased by 17–70% in button mushrooms and by 4–35% in crimini, when compared with the raw material. Moreover, the percentage participation of individual forms of vitamin B₆ changed. After 8 months of storage, pyridoxamine constituted 81–100% of the sum of vitamin B₆ in button mushrooms and 62–93% in crimini mushrooms. As for the participation of pyridoxine, it was 0% and 0–28%, respectively, and in the case of pyridoxal 0–18% and 4–38%, respectively. In comparison, Bernaś and Jaworska^[2] only found the presence of pyridoxamine and pyridoxal in frozen button mushrooms, and it should be specified that pyridoxamine constituted 80–100% of the sum of vitamin B₆. The change in the participation of different forms of vitamin B₆ in the total sum of this ingredient that was shown in the manuscript may well have been the result of the transformation of one form of vitamin B₆ into another. In addition, according to Leskova et al.,^[6] different forms of vitamin B₆ are characterized by different levels of stability as pyridoxal and pyridoxamine are more sensitive to heating, oxygen, and light than pyridoxine.

According to Jaworska et al.,^[36] as well as to Bernaś and Jaworska,^[2,21] a considerable effect on the quality of Agaricus bisporus during long-term frozen storage is caused by the preliminary treatment applied before freezing. Bernaś and Jaworska^[2] showed that of the vitamins soluble in water and in fats, considerable differences between frozen mushrooms were observed only in the case of vitamin B₃, B₆, and α-tocopherol. The preliminary treatment applied in the research also had an effect on the quantity of the vitamins from the B-group, although it must be specified that it depended on the kind of vitamin under analysis and on the variety of A. bisporus. In the case of button mushrooms, the highest amounts of vitamins B₁ and B₂ were found in unblanched frozen mushrooms, whereas in crimini it was found in frozen mushrooms that were unblanched (vitamin B₁) or blanched in water (vitamin B₂). In the case of button mushrooms, the highest loss of vitamins B₁ and B₂ was found in vacuum-impregnated frozen samples and in the case of crimini in fruiting bodies blanched in a sodium erythorbate and citric acid solution. As for vitamin B₆, it was difficult to point out any unambiguous tendencies. However, at the end of the frozen storage, in the case of both varieties, the highest loss was observed in unblanched mushrooms, with the lowest in mushrooms that had been vacuum impregnated (white button) or blanched in a sodium erythorbate and citric acid solution (crimini)

Conclusion

The varieties of Agaricus bisporus under analysis (white – button; brown – crimini) responded in different degrees to the preliminary treatment applied before freezing and frozen storage. With respect to the level of umami-taste active components, it may be stated that in the white variety of Agaricus bisporus there was a higher synthesis of 5’-nucleotides than in the brown variety – the synthesis of free amino acids. Out of all umami-taste active components, the main components responsible for the umami taste in the case of both varieties were 5’-TMP, 5’-UMP, and L-glutamic acid. The level on the EUC index (the equivalent of MSG) was higher in crimini mushrooms, mainly due to the higher levels of free amino acids, which was also confirmed in the sensory evaluation of taste. As for the level of the vitamins from the B-group, it may be stated that in the case of button mushrooms, the loss mainly regarded vitamin B₆ and in the case of crimini, vitamin B₂.

Additional information

Funding

The study was financed by the Agricultural University of Krakow under research Project No. BM-4737/KSiPOW/2013.