Aroma-enhancing role of Pichia manshurica isolated from Daqu in the brewing of Shanxi Aged Vinegar

Figures & data

Table 1. Eleven isolated yeast strains with good ester-producing performance.

Strains	Ester content (g/L)^a	Significance^b
		5% level	1% level
Y14	36.06 ± 0.48	a	A
Y2	33.76 ± 0.14	b	B
Y18	33.48 ± 0.22	b	B
Y12	32.90 ± 0.19	c	C
Y5	32.09 ± 0.26	d	D
Y6	29.67 ± 0.47	e	E
Y10	28.25 ± 0.64	f	F
Y11	27.15 ± 0.71	g	G
Y13	26.46 ± 0.59	h	H
Y9	23.71 ± 0.85	i	I
Y15	18.29 ± 0.40	j	J
Y21^c	11.16 ± 0.29	k	K

^aValues are expressed as means ± standard deviation.

^bMeans within the same column with different letters differ significantly.

^cY21 refers to the control ANGEL commercialized aroma-producing yeast used to enhance aroma during liquor or vinegar fermentation.

Table 2. Physiological and biochemical traits of Y14, later identified as Pichia manshurica.

Sugar fermentation
D-Glucose	+w^a	Maltose	–^a	Raffinose	–	lactose	–
Galactose	–
Carbon source assimilation (as sole carbon source)
D-mannitol	+^a	Succinic acid	+	Lactic acid	+	Glycerol	+w
Cellobiose	–	D-arabinose	–	Maltose	–	Ribitol	–
Trehalose	–	L-rhamnose	–	Raffinose	–	Inositol	–
D-gluconate	–	Butane-2,3-diol	–	Melibiose	–	Citric acid	–
D-xylose		Sucrose	–	D-ribose	–	Salicin	–
Starch	–	Erythritol	–	Melezitose	–	Lactose	–
Propane-1,2-diol	–	Myo-inositol	–	L-sorbose	–	D-Galactose	–
Nitrogen source assimilation (as sole nitrogen source)
Potassium nitrate	+	Sodium nitrite	+	Cadaverine	+	Glucosamine	+
L-lysine	+	Ethylamine	+	Creatinine	–	Imidazole	–
Other biochemical tests
Litmus milk test	–	Diazonium blue b reaction	–	Amyloid synthesis	–	Urease test	+

^a+: Positive; +W: weakly positive; –: negative

Figure 1. Vegetative cells (grown in liquid wart, 400× magnification by optical microscopy, left) and ascospores (stained green, blue, or colourless, right) of Pichia manshurica isolated from Daqu used for Shanxi Aged Vinegar brewing.

Figure 2. The distance tree of Y14 (highlighted in yellow) based on the sequence of KP027538.1.

Table 3. Peak area and aroma feature of volatile compounds from ethanol fermentation.

Types	Identified compounds^a	Aroma features	Area percentage(%)
			CF	FF
Alcohols	Ethanol	Spirit flavour and piquant	31.912	39.861
	Isoamyl alcohol	Apple-like, pungent	28.505	23.255
	Phenethyl alcohol	Rosy	0.255	0.327
	2-Methyl-1-butanol	Piquant and irritate	0.030	0.012
	Hexyl alcohol	Fruity fragrance	0.034	0.021
	1,2-Propanediol	Flavourless	ND^b	0.006
Esters	Ethyl acetate	Strong ether smell;clear and slight fruity, liquor fragrance	9.839	22.707
	Isoamyl acetate	Strong fresh fruity aroma	1.025	1.291
	Ethyl caproate	Flavour of pineapple and banana	0.679	0.721
	Ethyl octanoate	Rosy, aroma of orange flower and fruit	0.592	0.596
	Ethyl caprate	Wine fragrance	0.213	0.174
	Ethyl butyrate	Heavy, clear, and elegant fragrance of sweet fruit, pineapple, banana, and apple	0.195	0.077
	Isobutyl acetate	Smell of ripened fruit	0.113	0.073
	Ethyl heptanoate	Smell of fruit and wine, pleasant burnt taste	0.065	0.079
	Ethyl 2-methylbutyrate	Strong aroma of peels of apple, pineapple, and immature plum	0.032	0.083
	Ethyl benzoate	Slight fruity flavour	0.027	0.024
	Isovaleric acid ethyl ester	Apple-like and mulberry-like flavour	0.016	0.056
	Isopentyl hexanoate	Fragrance of apple and pineapple	0.016	0.014
	Ethyl palmitate	Weak fragrance of wax and butter fat	0.015	0.029
	Ethyl laurate	Peanut fragrance	0.013	0.013
	Benzoic acid methyl ester	Strong flowery and fruity aroma, powerful wintergreen oil taste	ND	0.025
Aldehyde	Acetal	Flowery, rosy, honey-like fragrance	0.513	0.326
	Benzaldehyde	Special almond fragrance	0.030	0.051
	Octanal	Fruity	0.009	0.011
	Nonanal	Rosy, flavour of oranges and tangerines, strong lipid taste	0.025	0.019
	Undecanal	Rosy, waxy, and fatty smell, sweet tangerine taste	0.016	0.010
	lauraldehyde	Violet fragrance at low concentration	0.016	0.021
	Decanal	Sweet, orange and tangerine like, waxy, flowery	0.015	0.015
	Pentadecanal	No description	ND	0.012
	Tetradecanal	Fatty, waxy, milk, butter fat, fish, fruity	ND	0.011
Others	2-Pentylfuran	Fruity aroma	0.168	0.140
	3-Octanone	Fruity aroma	ND	0.004
	2-Ethoxyethanol	Flavourless	ND	2.862

^aCompounds were detected and quantified by gas chromatography-mass spectrometry;

^bND means not detected.

Figure 3. Gas chromatography-mass spectrometry ion map (6–40 min) of Y14-fortified alcoholic fermentation (upper) and Daqu-alone alcoholic fermentation (bottom).

Table 4. The contents (g/L) of five types of basic compounds in the vinegar of the present study.

Vinegar	Total acid	Non-volatile acid	Total ester	Reducing sugar	Amino nitrogen
V1^a	37.4 ± 0.3^c	9.0 ± 0.6	15.30 ± 0.64	1.30 ± 0.7	1.53 ± 0.42
V2^b	38.0 ± 0.4**^d	7.9 ± 0.3**	21.54 ± 0.87**	12.5 ± 1.1*	1.46 ± 0.27**

^aV₁ refers to the vinegar using Daqu alone as starter.

^bV₂ refers to the vinegar using Daqu as starter and fortified with Y14 (P. manshurica).

^cData are expressed as means ± standard deviation.

^dValues marked with* and ** indicate significantly different from the control vinegar at the level of 5% and 1%, respectively.

Table 5. Sensory evaluation of Y14-fortified Daqu vinegar versus Daqu-alone vinegar.

Vinegar^a	Ester	Sour	intensity	Fruity	Flowery	Sweet	Aftertaste
V1	20^b	42	20	2	2	13	20
V2	32	45	18	12	8	12	26

^aV₁ and V₂ represent vinegar from Daqu-based fermentation and Y14-fortified Daqu-based fermentation, respectively.

^bAverage flavour scores (on a scale of 0–100) presented by eight taste panel members