Effect of rose polyphenols on oxidation, biogenic amines and microbial diversity in naturally dry fermented sausages

Accepted Manuscript Effect of rose polyphenols on oxidation, biogenic amines and microbial diversity in naturally dry fermented sausages Qiu Qin Zhang, Mei Jiang, Xin Rui, Wei Li, Xiao Hong Chen, Ming Sheng Dong PII:

S0956-7135(17)30100-7

DOI:

10.1016/j.foodcont.2017.02.054

Reference:

JFCO 5489

To appear in:

Food Control

Received Date: 4 November 2016 Revised Date:

26 January 2017

Accepted Date: 21 February 2017

Please cite this article as: Zhang Q.Q., Jiang M., Rui X., Li W., Chen X.H. & Dong M.S., Effect of rose polyphenols on oxidation, biogenic amines and microbial diversity in naturally dry fermented sausages, Food Control (2017), doi: 10.1016/j.foodcont.2017.02.054. 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.

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Effect of rose polyphenols on oxidation, biogenic amines and microbial diversity

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in naturally dry fermented sausages

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Running Title: Application of rose polyphenols in sausages

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Qiu Qin Zhanga, Mei Jianga, Xin Ruia, Wei Lia, Xiao Hong Chena, Ming Sheng

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Donga b*

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College of Food Science and Technology, Nanjing Agricultural University,

Nanjing 210095, PR China b

Jiangsu Collaborative Innovation Center of Meat Production and Processing,

Quality and Safety Control

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*Corresponding

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[email protected]

Author:

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ACCEPTED MANUSCRIPT Abstract

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Rose polyphenols (RPs) have antioxidant and antibacterial activities. In oreder to

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evaluated the application of RPs in meat products, the effect of RPs on the safety of

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fermented sausages was investigated. In the present study, RPs inhibited the increase

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of pH. Phenylethylamine, putrescine, cadaverine, tyramine and spermidine can be

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detected in sausages, while spermine and tryptamine were not observed. Sausages

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containing RPs had lower thiobarbituric acid reactive substance values and biogenic

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amines than the control. Moreover, RPs decreased the total bacterial count but

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increased growth rate of lactic acid bacteria. The effect of RPs on oxidation, biogenic

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amines and bacterial growth is dependent on the concentration. High-throughput

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DNA sequence analysis revealed that operational taxonomical units (OTUs) of

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samples without PRs were higher than those contaning 3 mg/g RPs, and 323 OTUs

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shared between two batches. Especially, a higher level of Lactobacillales OTUs was

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observed in the 3 mg/g batches. These results indicated that RPs could be used to

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improve the safety of fermented sausage by inhibiting lipid oxidation, biogenic

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amines formation and spoilage bacterial growth, and increasing growth rate and

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richness of lactic acid bacteria.

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Keywords

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Sausage, Polyphenol, Oxidation, Biogenic amines, Microbial diversity

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ACCEPTED MANUSCRIPT 1. Introduction

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Naturally dry fermented sausages are appreciated by consumers because of their

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special flavor and texture characteristics. In China, consumption of traditional

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dry-fermented sausages, knows as "Lachang", exceeds eight million tons annually

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(Ahmad, 2014). Traditionally, naturally dry fermented sausages were directly kept in a

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cool and ventilated place to gradually dry up and produce a typical flavor and texture

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(Ahmad, 2014, Toldrá et al., 2008). For several decades, this kind of fermented

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sausages is produced directly in temperature- and humidity-controlled dryers with a

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long fermentation period (Aquilanti et al., 2007; Latorre-Moratalla et al., 2008).

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Major chemical and microbiological quality usually changes during this period. The

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safety of naturally dry fermented sausages were harder to control, because they are

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made without starter cultures and relies entirely on bacteria present in meat and in

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surrounding microflora. Lipid oxidation, a common reaction in fermented sausages,

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changes meat quality parameters, such as colour, flavour, odour, texture and even the

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nutritional value (Chaves-Lopez et al., 2015; Jin et al., 2015; Nowak et al., 2016;

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Siripatrawan and Noipha, 2012). Oxidation is a major cause of quality deterioration

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and often limits the shelf life of fermented sausages (Talon et al., 2008). Besides

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oxidation, biogenic amine formation also occurs in meat products. Some biogenic

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amines, such as tyramine and histamine have the vasoactive and psychoactive effects.

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Biogenic amines have been proposed as possible indicators of the unsafety of meat

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productes (Latorre-Moratalla et al., 2008). In fermented sausages, biogenic amines

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fromation are mainly related to the presence of microorganisms (Lu et al., 2015;

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Parente et al., 2001). Moreover, the quality of fermented sausages is related to the

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microbial diversity. Therefore, natural antioxidant and antibacterial substances are

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able to affect the quality and safety of fermented sausages.

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Polyphenols, one of the most numerous and ubiquitous group of plant secondary

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metabolites, have been widely extracted from plant, such as mint (Latorre-Moratalla

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et al., 2008), tea (Fan et al., 2014), leafy green vegetable (Tagliazucchi et al., 2010),

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cherry and blackcurrant leaves (Nowak et al., 2016). Polyphenols can exhibit

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antiallergenic, antiinflammatory, antioxidant (Aquilano et al., 2008; Tagliazucchi et

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al., 2010; Lorenzo et al., 2014), antibiofilm (Zhang et al., 2015) and antibacterial

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activities (Delgado et al., 2012; Radha et al., 2014). In meat products, polyphenols

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have been proved to be effective against pathogens or spoilage organisms (Nowak et 3

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al., 2016; Siripatrawan and Noipha, 2012), oxidation reactions (Demir et al., 2014;

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Latorre-Moratalla et al., 2008), and biogenic amine formation (Fan et al., 2015; Wang

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et al., 2015). Hence, natural polyphenols, as a sources of antioxidants and

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antibacterial agent, have potential application in meat products. Rose, commonly used as raw food material, contains a high content of

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polyphenols. Rose polyphenols (RPs) have been proved to have antioxidants (Demir

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et al., 2014), antiswarming and antibiofilm activities (Zhang et al., 2015). However,

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few studies have been carried out on the application of RPs in meat products.

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Therefore, the present study investigated the effect of RPs on the pH, oxidation,

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biogenic amines, and microbiology of fermented sausages.

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2. Materials and methods

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2.1 Preparation of rose polyphenols (RPs)

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Rose polyphenols (RPs, Jinwawa Company, Jiaxing, China) were prepared as

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previously described (Zhang et al., 2015). Briefly, RPs was extracted by 80 W

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ultrasound at 50 oC for 30 min in 10-fold water, purified by macroporous resin

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HPD600 (Qinshi Technology, Ltd., Zhengzhou, China), dried under vacuum at 45 oC,

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and stored at -20 oC. The total polyphenols of RPs was 111.09 mg gallic acid

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equivalents per g dry weight.

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2.2 Preparation of fermented sausages

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Fermented sausages were manufactured according to the method reported by

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(Rai et al., 2010), with a slight modification. Nine point six kilogram of lean pork and

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two point four kilogram of backfat (Sushi Group, Jiangsu, China) were diced into

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pieces using a meat grinder (MG815S, Chulux Company, Shenzhen, China), and

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equally divided into a control without RPs and 3 batches supplemented with 1, 2 and

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3 mg RPs per g of meat. Ground meat was mixed with ingrediends as follows: 80%

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lean pork (w/w, Sushi Group, Jiangsu, China), 20% pork backfat (w/w, Sushi Group,

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Jiangsu, China), 2% salt (w/w), 2% sugar (w/w), 1% rice wine (w/w), 0.05% sodium

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erythorbate (w/w), 0.2% sodium polyphosphate (w/w), 0.5% sodium glutamate (w/w),

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0.012% sodium nitrite (w/w) and without any starters. Ingredients and meat were

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blended in a bowl mixer (JF330L, Shengli Company, Ningbo, China) for 3 min. The

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mixtures were stuffed into hog casings (Far-Eastern Casing Company, Hebei, China)

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with a diameter of approximately 5 cm, linked into a length of 8-10 cm and a weight

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ACCEPTED MANUSCRIPT of 110 ± 10 gram. Each batch contained 30 sausages. The sausages were fermented

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and dried in an incubator (LHS150SC, Hengke Company, Shanghai, China) at 20 oC

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and 90% relative humidity (RH) for 3 days, 10 oC and 80% RH for 5 days, and 10 oC

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and 70% RH for 16 days. Three sausages of each batch were taken randomly for

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chemical and microbiological analysis at 0, 3, 8, 15, 24 d. This experiment was

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repeated three times.

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2.3 The water activity and moisture determination

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Water activity was determined using an water activity apparatus (AW-1, Bibo

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Electronic Apparatus Inc., Wuxi, China) as previously described (Li et al., 2013).

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Moisture was determined as previously described (Rubilar et al., 2013). Five grams of

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sample dried at 105 oC to constant weight. The sample was weighted before and after

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drying. The moisture was calculated and expressed as the difference between the

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weights before and after drying.

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2.4 Determination of pH

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The pH value was evaluated according to the procedures developed by Lu et al.

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(2015). Five grams of sample was homogenized in 20 ml of deionized water for 5 min

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using an Ultra-turrax model T25 basic homogenizer (IKA Company, Staufen,

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Germany). The pH was measured using a digital pH meter (PHS-4A, Jingke Company,

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Shanghai, China) at room temperature.

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2.5 Determination of thiobarbituric acid reactive substance (TBARS) values

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The thiobarbituric acid reactive substance (TBARS) was determined according

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to the method of Wang et al. (2016) with some modifications. Ten grams of

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homogenized sausage sample was extracted twice with 25 ml of 7.5% (w/v)

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trichloroacetic acid. The extracts were centrifuged at 2000 g for 10 min. Then 5 ml of

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supernatant was mixed with 5 ml 0.02 M thibarbituric acid reagent. The mixture

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heated in 95 oC water bath for 40 min. After cooling, the absorbance was measured at

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532 nm. The TBARS value was expressed as mg malonaldehyde (MDA)/kg sample.

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2.6 Biogenic amine determination

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Histamine, putrescine, cadaverine, phenylethylamine, tryptamine, tyramine,

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spermidine, and spermine standards were purchased from Sigma. Standard solutions 5

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perchloric acid and stored at 4 oC. Biogenic amines determination was carried out

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according to the procedures developed by Mozuriene et al. (2016) with some

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modifications. Five grams of sample was homogenized (Ultra-Turrax T25, IKA

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Company, Staufen, Germany) in 20 ml of 0.4 M perchloric acid for 5 min, centrifuged

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at 5000 g for 10 min (Allegra X-22, USA). The supernatants were collected and

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adjusted to 50 ml with 0.4 M perchloric acid. One milliliter of the extract or standard

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solution was mixed with 200 µl of 2 M sodium hydroxide, 300 µl of saturated sodium

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bicarbonate and 1 ml of 10 mg/ml 5-(dimethylamino) naphthalene-1-sulfonyl chloride

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(dansyl chloride reagent). After incubation at 40 oC for 30 min, the mixture mixed 100

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µl of 25% ammonium hydroxide and adjusted to 5 ml with acetonitrile. Finally, the

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mixture was filtered through a 0.4 µm filter for high performance liquid

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chromatography (HPLC) analysis.

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HPLC analysis was carried out with an Eclipse plus C18 guard column (5 µm, 4.6

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mm × 250 mm, Agilent, USA) at 254 nm. The elution was performed with acetonitrile

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(solvent A) and water (solvent B) using the following gradient elution program for

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separation: 0-2 min, 55-65% acetonitrile; 2-4 min, 65%-70% acetonitrile; 4-10 min,

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70% acetonitrile; 10-17 min, 70-90% acetonitrile; 17-18 min, 90-100% acetonitrile.

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The flow rate was 0.8 ml/min.

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2.7 Microbiological counts

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Ten grams of sample was homogenized in 90 ml of sterile saline-peptone water

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(SPW, containing 0.85% NaCl and 0.1% peptone) for 2 min using a laboratory

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stomacher (HN-08, Hannuo Company, Shanghai, China). The homogenate was

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collected and serially diluted in triplicate (1:10) in SPW. The samples were

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spread-plated on Plate Count Agar (PCA, Lu Qiao Company, Beijing, China) for total

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viable counts, Man Rogosa Sharpe Agar (MRS, Lu Qiao Company, Beijing, China)

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for lactic acid bacteria counts. All plates incubated at 37 oC for 24 h and then

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examined typical colony.

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2.8 DNA extractions and 16S rDNA sequencing

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At the end of fermentation, DNA was extracted from sausages containing 0 and 3

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mg/g RPs according the method reported by Lu et al. (2015), with a slight

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modification. Five gram of sample was homogenized in 20 ml of deionized water for 6

ACCEPTED MANUSCRIPT 10 min using a laboratory stomacher (HN-08, Hannuo Company, Shanghai, China).

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Lipid was removed from sample by centrifugation at 12000 g for 30 min at 4 oC. The

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sample was rehomogenized using a homogenizer (BME 100LX, Shanghai Weiyu Co.,

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China) under aseptic conditions, and centrifuged at 500 g for 10 min at 4 oC. The

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supermentant was transferred to sterile centrifuge tubes, and centrifuged at 10000 g

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for 10 min at 4 oC. The pellet was dissolved in 3 ml sterile water, and mixed with an

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equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). After centrifugation at

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12000 g for 10 min at 4 oC, the supermentant was used to DNA extractions using the

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E.Z.N.A. Genomic DNA Kit (Omega bio-tek, Inc., USA) according to manufacturer’s

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instructions. The total DNA was eluted in 50 µl of Qiagen elution buffer, and stored at

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-80 oC. DNA extraction of each samples were generated for two replicates. Primers

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319F

(5'-ACTCCTACGGGAGGCAGCAG-3')

and

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(5'-GGACTACHVGGGTWTCTAAT-3') were used to amplify the V3-V4 region of

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bacterial 16S rDNA. PCR reactions were carried out in a total volume of 25 µl

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containing 25 ng genomic DNA extract, 12.5 µl PCR Premix and 2.5 µl of each

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primer. Amplifications were performed on an Eppendorf Mastercycler gradient

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thermocycler (Eppendorf, Hamburg, Germany) using the following program:

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denaturation at 98 oC for 30 s, 35 cycles of denaturation at 98 oC for 10 s, annealing at

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54 oC for 30 s, extension at 72 oC for 45 s, and final extension at 72 oC for 10 min.

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PCR products were analyzed in 2% agarose gel electrophoresis. Each sample was

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measured in duplicates. PCR products from the same batch were combined together

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and normalized by AxyPrep TM Mag PCR Normalizer (Axygen Biosciences, Union

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City, CA, USA). The amplicon pools were prepared for sequencing with AMPure XT

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beads (Beckman Coulter Genomics, Danvers, MA, USA) and the size and quantity of

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the amplicon library were assessed on the LabChip GX (Perkin Elmer, Waltham, MA,

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USA) and with the Library Quantification Kit for Illumina (Kapa Biosciences,

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Woburn, MA, USA), respectively. PhiX Control library (v3) (Illumina) was combined

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with the amplicon library (expected at 30%). The libraries were sequenced either on

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300PE MiSeq runs and one library was sequenced with both protocols using the

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standard Illumina sequencing primers, eliminating the need for a third index read.

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The raw sequence data were analyzed using the Quantitative Insights Into

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Microbial Ecology (QIIME). Low-quality sequences with an average quality score of

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less than 10 were removed. After denoising the sequences using Denoiser 0.91, and

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checking for chimeras using Usearch v6.0.307. A total of 45736 and 26387 clean 7

ACCEPTED MANUSCRIPT sequences were obtained from the control and 3 mg/g batch, respectively. Ninety-nine

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percent of resulting sequences was ranged from 400 to 500 bp. The CD-HIT pipeline

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was used for picking operational taxonomic units (OTUs) through making OTU table.

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Sequences were assigned to OTUs at 97% similarity. Representative sequences were

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chosen for each OTU, and taxonomic data were then assigned to each representative

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sequence using the Ribosomal Database Project (RDP) classifier. Alpha diversity

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caculation was performed based on the rarefied OTU table to compare the diversity of

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two samples. Chao 1 richness, Simpson, Shannon diversity and ennness indexes were

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determined with the vegan package in R (The R Foundation for Statistical Computing,

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Vienna, Austria). Taxa phylogeny was drawn with Graphical Phylogenetic Analysis

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(GraPhlAn, http://huttenhower.sph.harvard.edu/GraPhlAn).

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2.9 Statistical analysis

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All data were reported as means ± standard deviation of triplicates. To determine

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if there were any significant differences by the effect of the RPs, statistical analysis

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was performed by two-way analysis of variance (ANOVA) with Tukey test using the

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Statistica software (version 5.0, StatSoft. Inc., Tulsa, USA). Differences with P values

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< 0.05 were considered statistically significant.

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3. Results and discussion

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The traditional sausage was made without starter cultures and relies entirely on

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bacteria present in meat and in surrounding microflora. During the fermentation, no

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significant difference (P > 0.05) in moisture and water activity was observed among

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the four batches, in agreement with the study of Li et al.(2013), who found

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polyphenols from green tea or grape seed had no effect on the moisture changes of

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dry-cured sausages. The moisture and water acitiviy of finial products decreased to

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approximately 30% and 0.80, respectively. The 24-day fermentention was similar to

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values of traditional dry-fermented sausage without starters (Rai et al., 2010; Li et al.,

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2013), but shorter than values of industrial fermented sausages with starters (Komprda

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et al., 2001; Hughes et al., 2002; Fernández-López et al., 2008). The difference was

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mainly due to the fact that low temperature and humidity were used to inhibite growth

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of spoilage bacteria (Rai et al., 2010).

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Fig. 1 showed the pH changes of sausages during fermentation. During

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fermentation the pH of fermented sausage decreased slightly followed by gradual 8

ACCEPTED MANUSCRIPT increases. Similar trends in pH were observed in study by Li et al.(2013). The final

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pH was ranged from 6.34 to 6.45, similar to the value reported by Li et al.(2013), but

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higher than values reported by Sun et al., (2016) and Chaves-Lopez et al. (2015). The

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difference was due to the population of lactic acid bacteria in sausages was different

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(Leroy and De Vuyst, 2004; Liu et al., 2011). At the same fermentation time, the pH

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of the control was similar to the 1mg/g batch, and significantly higher (P < 0.05) than

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2 and 3 mg/g batches. This is mainly contributed to the effect of polyphenols on the

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growth of ammonia-producing bacteria (Flythe et al., 2010).

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Fig.2 showed the effect of RPs on the TBARS value of fermented sausages

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during fermentation. The TBARS values of control samples increased from 0.51 to

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6.04 mg MDA/kg. Zhang et al. (2016) reported that the range of TBARS values for

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pork loin was 0.175 to 0.664 mg MDA/kg during 4 oC storage for 9 days. Compare

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with pork loin, TBARS value of sausages was higher and increased faster. This is due

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to the high fat content (20%) and fermented temperature (10 oC). The increase of

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TBARS, commonly observed in meat products, mainly due to the lipid oxidation

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(Pelser et al., 2007; Zanardi et al., 2004). Fan et al.(2015) indicated that besides lipid

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oxidation, the increase in TBARS values was also attributed to the dehydration of

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samples. From 3 to 24 d, TBARS values were significantly lower (P < 0.05) in

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batches with higher RPs content. This result suggested that RPs had hight antioxidant

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activities and exhibited an inhibitive effect on oxidation. Moreover, the antioxidation

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activity of RPs is dependent on the concentration. Plant extracts, such as herb (Shan et

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al., 2009), tea (Bozkurt, 2006; Lorenzo et al., 2014), grape (Li et al. 2013; Lorenzo et

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al., 2014), blackcurrant (Nowak et al., 2016), canola (Brettonnet et al., 2010) and

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olive (De Jong and Lanari, 2009), are able to prevent lipid oxidation in food

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emulsions, due to the contribution of polyphenol compounds. Thus, RPs can be

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applied as a source of natural antioxidant in meat products.

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Six biogenic amines (phenylethylamine, putrescine, tyramine, cadaverine,

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spermidine and histamine) were detected from sausages, while spermine and

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tryptamine can not be observed (Fig. 3). This result was similar to the study of Lu et

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al. (2010), who found the concentration of spermine and tryptamine in fermented

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sausages were lower than other biogenic amines. Biogenic amines increased with the

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fermentation time, due to the decarboxylation of amino acids (Suzzi and Gardini,

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2003). At the end of the fermentation, the concentration of biogenic amines were in

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the following order: phenylethylamine > spermidine > cadaverine > tyramine > 9

ACCEPTED MANUSCRIPT histamine > putrescine. At the same fermentation time, the highest biogenic amines

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concentration was observed in the control, while the lowest was in the batch

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supplemented with 3 mg/g RPs. Obviously, RPs inhibited the biogenic amines

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formation in sausages. Formation of biogenic amines are associated with growth of

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bacteria (Durlu-Özkaya et al., 2001; Lázaro et al., 2015). Provious study confirmed

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that inhibitive effect on biogenic amines fromation was due to the antibacterial

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activity of poyphenols (Bozkurt, 2006; Fan et al., 2015; Wang et al., 2015).

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The microbiological counts of sausages during fermentation were presented in

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Fig. 4. The total bacteria and lactic acid bacteria of the control increased from 3.13 to

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6.32 log cfu/g and from 2.75 to 4.83 log cfu/g, respectively. Since no starter was used,

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the initial population of lactic acid bacteria in traditional fermented sausages is lower

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than that in industrial fermented sausages (Ikonic et al., 2015). From 15 to 24 d, the

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control had the highest total bacterial count. However, lactic acid bacteria in sausages

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containing RPs were significantly (P < 0.05) higher than that in control from 0 to 10 d.

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Therefore, RPs increased growth rate of lactic acid bacteria, but inhibited the

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increasing of total bacteria. This may been due to the fact that the sensitivity of

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bacteria to polyphenols varied from species to species (Nowak et al., 2016).

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Since the chemical characteristics and microbiological count of the control and 3

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mg/g batches were significantly different, the present study detected the microbial

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community profiling of these two batches at day 24 of fermentation. Microbial

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richness parameters (Table 1) indicated that the bacterial community was more

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diverse in the control than in batch supplemented with 3 mg/g RPs. The Simpson

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index of these two batches were same, suggesting that the bacterial evenness were

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similar. According to a study by Zhang et al. (2015), RPs had a quorum-sensing

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inhibitive effect on gram-negative bacteria. Given the quorum quenching, the ability

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of gram-negative bacteria to adapt to environment may be reduced, and the bacterial

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diversity decreased. The control and 3 mg/g batches had similar dominant species

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which

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Staphylococcus and Kocuria (Fig. 6). However, the OTUs of Lactobacillales in the 3

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mg/g batch was higher than that in control batch, suggesting RPs increased the

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richness of Lactobacillales. These results are in agreement with previous studies

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indicating that the presence of grape polyphenols can promote growth of lactic acid

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bacteria (Tabasco et al., 2011; Zhao and Shah, 2014). Since strains belonging to

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Lactobacillales usually added as starter cultures, addition of RPs could influence the

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were

identified

as

Pseudomonas,

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Psychrobacter,

Acinetobacter,

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microbial security and safety of fermented sausage. 4. Conclusions

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Rose polyphenols (RPs) can prevent the increase of pH, lipid oxidation and

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biogenic amine formation. Moreover, RPs decreased total bacteria and bacterial

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diversity. However, RPs can selectively modify the growth of lactic acid bacteria by

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increased growth rate and richness. Therefore, RPs were effective to improve the

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safety of fermented sausage.

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Acknowledgments

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This study was co-financed by Youth Science Fund Project of the National

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Natural Science Foundation of China (31601494), and Fundamental Research Funds

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for the Central Universities (KYZ201544 and KJQN201729). All authors declare that

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they have no conflict of interest.

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Supporting Information

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Additional Supporting Information may be found in the online version of this

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article: Fig. S1 The moisture and water activity (Aw) of sausages during fermentation. Error bars represent the standard deviations of mean values. Fig. S2 Rank abundance curves of the bacterial 16S rDNA sequences.

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Table S1 The length distribution of the bacterial 16S rDNA sequences.

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Table S2 Quality scores of the bacterial 16S rDNA sequences.

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Table 1 Diversity indices for the 16S rDNA sequences of sausages without rose polyphenols or containing 3 mg/g rose polyphenols on 24 d.

Chao1

Shannon

Simpson

2782 1704

25927.72 14223.04

4.02 3.52

0.68 0.68

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Observed

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OUT richness

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Figure Legends

Fig. 1. Effect of rose polyphenols on the pH values of fermented sausages. Error bars represent the standard deviations

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time. NS indicates no significant difference at the 0.05 level.

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of mean values. a-b: Different letters above bars indicate significant differences between the various treatments at the same

Fig. 2. Effect of rose polyphenols on the TBARS values of fermented sausages. Error bars represent the standard deviations of mean values. a-d: Different letters above bars indicate significant differences between the various treatments at

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the same time. NS indicates no significant difference at the 0.05 level.

Fig. 3. The effect of rose polyphenols on the biogenic amines formation in fermented sausages. a-c: Different letters

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difference at the 0.05 level.

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above bars indicate significant differences between the various treatments at same time. NS indicates no significant

Fig. 4. Effect of rose polyphenols on the growth of total bacteria (A) and lactic acid bacteria (B). Error bars represent the standard deviations of mean values. a-c: Different letters above bars indicate significant differences between the various

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treatments at same time. NS indicates no significant difference at the 0.05 level.

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Fig. 5. The phylogenetic relationships of bacteria in the final sausages without rose polyphenols (A) and containing 3

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mg/g rose polyphenols (B) at 24 d.

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Highlights 1. The moisture reduction of fermented sausages was affected by fermentation time, but not by rose polyphenols. 2. Addition of rose polyphenols inhibited lipid oxidation and biogenic amine formation. 3. Rose polyphenols can selectively modify the growth rate and richness of lactic acid bacteria.